- Email: [email protected]
AChR-myasthenia gravis switching to double-seropositive several years after the onset
Journal of Neuroimmunology 267 (2014) 111–112
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
AChR-myasthenia gravis switching to double-seropositive several years after the onset Vasiliki Zouvelou a,⁎, Paraskevi Zisimopoulou b, Erasmia Psimenou c, Eirini Matsigkou b, Eleftherios Stamboulis a, Socrates J. Tzartos b,d a
Neurology Department, Aeginition Hospital, University of Athens, Greece Department of Biochemistry, Hellenic Pasteur Institute, Greece Renal Unit, Alexandra Hospital, Athens, Greece d Tzartos NeuroDiagnostics, Athens, Greece b c
a r t i c l e
i n f o
Article history: Received 29 September 2013 Received in revised form 8 December 2013 Accepted 17 December 2013 Keywords: Myasthenia gravis Acetylcholine receptor Muscle-specific kinase Double seropositive Cell-based assay Biphasic course
a b s t r a c t We report an early onset AChR-myasthenia gravis (MG) with biphasic clinical course. The clinical “switch” from AChR-MG to MuSK-MG emerged 16 years after the onset and 11 years after thymectomy. MuSK antibodies were detected only by cell-based assay and only upon clinical “switch”, while AChR antibodies remained positive and at high titers during the whole disease course. Although the occurrence of AChR antibodies and MuSK antibodies in the same individual is rare, the re-assessment of the antibody status, using all available assays, is advisable when there is clinical indication. © 2013 Elsevier B.V. All rights reserved.
1. Introduction
2. Case report
Antibodies to muscle-specific tyrosine kinase (MuSK Abs) are detected in approximately 40% of generalized acetylcholine receptor (AChR) antibody-negative myasthenia gravis (MG). MuSK-MG is a distinct clinical entity and its phenotype differs from AChR-MG. Occurrence of AChR antibodies (Abs) and MuSK Abs in the same individual is rare. The limited references concern double seropositivity detected at the onset of the disease (Zouvelou et al., 2013), years after the onset (Suhail et al., 2010) and “seroconversion” from AChR Abs to MuSK Abs taking place after thymectomy (Saulat et al., 2007; Sanders and Juel, 2008; Kostera-Pruszczyk and Kwiecinski, 2009). Routine testing for double seropositivity is not recommended in clinical practice, unless there is clinical indication. We report a female patient with AChR-MG which became double seropositive-MG many years after the onset. The reason for the re-assessment of antibody status was the biphasic clinical course which clearly indicated an immunological peculiarity.
A 36-year old Caucasian female with AChR-MG first came to our attention on January 2013. The onset of the disease was 17 years ago (1996), at the age of 19, when the presenting symptoms were diplopia and unilateral eyelid ptosis. Three months later MG generalized with upper and lower limbs involvement. Serum AChR Abs were positive at a high titer of 270 nM (positive ≥ 0.6 nM). She received only pyridostigmine for the first 5 years and in 2001 prednisolone was added to the regimen. On December 2001, she underwent thymectomy and the histological examination revealed follicular hyperplasia. In 2003 and in 2008 the patient gave birth to two offsprings suffering both from transient neonatal MG. At those periods the patient's titer of AChR Abs was 370 nM and 587 nM, respectively. From 1996 until 2012, the myasthenic weakness was restricted to ocular and limb muscles, well controlled with pyridostigmine and prednisolone and there was no episode of myasthenic crisis or requirement for short term immunotherapy i.e. plasma exchange (PE) or intravenous immunoglobulin. On May 2012, 16 years after the onset of MG, an unprovoked and severe clinical deterioration supervened which was characterized by prominent bulbar, facial, neck and respiratory muscle involvement with dramatic response to PE. The above-mentioned muscles had never been previously involved during the disease course. The patient received high dose prednisolone and cyclosporine (200 mg/d) and she remained on monthly courses of PE until the end of November 2012. At our first evaluation (early January 2013) the patient was in minimal
⁎ Corresponding author at: Department of Neurology, University of Athens, 74 Vas. Sophias Ave., Athens 11528, Greece. Tel.: +30 2107289404; fax: +30 2107216474. E-mail address: [email protected] (V. Zouvelou). 0165-5728/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jneuroim.2013.12.012
112
V. Zouvelou et al. / Journal of Neuroimmunology 267 (2014) 111–112
manifestations, as she reported no symptoms and the neurological examination revealed only mild fatigable lower limbs. MRI of the mediastinum did not show any residual thymic tissue. We considered that the last reported pattern of myasthenic weakness was unusual for longstanding and previously stable AChR-MG and highly indicative of MuSK-MG. For that reason we asked for re-assessment of antibody status. AChR Abs were positive at a titer of 366 nM, i.e. similar to her previous titers, while testing for MuSK Abs by a commercial radioimmunoprecipitation assay (RSR Ltd, Cardiff) and by an in house radioimmunoprecipitation assay (Trakas et al., 2011) was negative. However, a live Cell Based Assay (CBA) for MuSK Abs was then performed (according to Leite et al., 2008). Interestingly, the recent serum was found anti-MuSK positive by the CBA (at both test dilutions: 1:10 and 1:20), whereas sera derived during 2008, were found negative even at 1:10 dilution. At the end of January 2013, the patient reported for the first time asymmetric distal weakness of the hand, especially of the finger extensors, interfering with daily activities. This new pattern of weakness was directly attributed to MG, based on the findings of appropriate electrophysiological testing. Prednisolone was increased up to 60 mg/d with mild improvement of the distal weakness. While she was on high-dose prednisolone an exacerbation came up with moderate to severe bulbar and respiratory symptoms which responded to courses of PE. However, the distal weakness remained unaffected to the PE and it was the prominent disabling symptom until the last evaluation on September 2013. 3. Discussion The clinical course of our patient until May 2012 was rather typical of early onset AChR-MG. Later on, the pattern of the disease was more typical of MuSK-MG, as evidenced by the distribution of myasthenic weakness and the unprovoked exacerbations. The phenotype of MuSK-MG appears to be clinically distinct with individuals generally exhibiting prominent oculobulbar, neck and respiratory muscle involvement. Furthermore, exacerbations are common and can occur in the absence of triggering factors and despite high dose immunosuppression (Guptill et al., 2011). The finger extensor weakness of our patient emerged during the last year, it greatly interferes with daily activities and is refractory to immunotherapy, even PE. Although weakness of distal extremity muscles is unusual in MG, the propensity for finger extensor weakness has been described in the limited references of distal weakness in MG (Nations et al., 1999). Whereas repetitive nerve stimulation of distal muscles is normal in many MG patients, single-fiber electromyography (SF-EMG) of the extensor digitorum communis (EDC) muscle reveals excessive jitter in the majority of generalized MG patients (Oh et al., 1992). This finding emphasizes that neuromuscular transmission is impaired in the finger extensors of many MG patients, even if there is no clinical weakness. Distal weakness is highly unusual finding in MuSK-MG. However, in some of these patients an abnormal jitter in the EDC muscle can be found on SF-EMG (Stickler et al., 2005). In light of the biphasic clinical course of our patient, serological reassessment was undertaken which revealed the presence of both AChR Abs and MuSK Abs. Interestingly, the titer of AChR Abs remained high during the whole course, despite the immunotherapy, the thymectomy and the radiological absence of residual thymic tissue. MuSK Abs were not detected by the classical radioimmunoprecipitation assay but only by the novel CBA assay, which is known to detect MuSK Abs in several previously MuSK Ab negative patients (Vincent et al., 2012). This may suggest that MuSK CBA should be applied in both AChR Ab positive and negative patients with MuSK phenotype. The occurrence of both AChR Abs and MuSK Abs in the same individual is rare. We have recently reported a double seropositive case at the onset of MG and prior to any therapeutic intervention. The patient was a 36-year old woman with oculobulbar distribution of myasthenic weakness, responder to pyridostigmine and prednisolone and with low titer
of AChRAbs (Zouvelou et al., 2013). A double-seropositive MG patient has also been identified 8 years after the onset of the disease and 6 years after thymectomy. The antibody status at the onset or prior to thymectomy was not known (Suhail et al., 2010). Another case of double-seropositive MG patient was identified after administration of D-penicillamine, where both antibodies disappeared after D-penicillamine discontinuation (Poulas et al., 2012). Furthermore, there are three reports of childhood onset AChR-MG who “seroconverted” to MuSK-MG after thymectomy, possibly as a result of “epitope spread”. None had MuSK Ab titers checked at the onset and all had relatively low AChR Abs (Saulat et al., 2007; Sanders and Juel, 2008; KosteraPruszczyk and Kwiecinski, 2009). Recently, a case of clinically biphasic MG with both AChR and MuSK Abs has been reported (Rajakulendran et al., 2012). The onset was at age 13 with bulbar predilection of the myasthenic weakness. The patient underwent thymectomy and responded favorably for over 15 years, when exacerbations started to occur. In contrast to our case, AChR or MuSK Abs had never been tested before clinical “switch” i.e. at the onset, before thymectomy or during the clinical stable state. We have documented that our patient was AChR-positive at the onset, prior to thymectomy and after thymectomy till the “switch” of the phenotypic pattern. Indeed, the double seropositivity was detected upon clinical “switch” from a predominantly AChR-MG to a MuSK-MG. We excluded the possibility of its previous existence as sera derived during 2008 were found negative for MuSK antibodies tested both by radioimmunoprecipitation assay and CBA. The development of MuSK Abs many years after the disease onset and thymectomy as well the persistence of high titers of AChR Abs is intriguing. A plausible explanation for the emergence of MuSK Abs would be the immunological changes following thymectomy in AChR-MG. In particular, these immunological changes consist in an expansion in the T cell repertoire and a greater prevalence of autoantibodies and autoimmune diseases than in non-thymectomy cases (Gerli et al., 1999).
References Gerli, R., Paganelli, R., Cossarizza, A., Muscat, C., Piccolo, G., Barbieri, D., Mariotti, S., Monti, D., Bistoni, O., Raiola, E., Venanzi, F.M., Bertotto, A., Franceschi, C., 1999. Long-term immunologic effects of thymectomy in patients with myasthenia gravis. J. Allergy Clin. Immunol. 103, 865–872. Guptill, J.T., Sanders, D.B., Evoli, A., 2011. Anti-MuSK antibody myasthenia gravis: clinical findings and response to treatment in two large cohorts. Muscle Nerve 44, 36–40. Kostera-Pruszczyk, A., Kwiecinski, H., 2009. Juvenile seropositive myasthenia gravis with anti-MuSK antibody after thymectomy. J. Neurol. 256, 1780–1781. Leite, M.I., Jacob, S., Viegas, S., Cossins, J., Clover, L., Morgan, B.P., Beeson, D., Willcox, N., Vincent, A., 2008. IgG1 antibodies to acetylcholine receptors in “seronegative” myasthenia gravis. Brain 131, 1940–1952. Nations, S.P., Wolfe, G.I., Amato, A.A., Jackson, C.E., Bryan, W.W., Barohn, R.J., 1999. Distal myasthenia gravis. Neurology 52, 632–634. Oh, S.J., Kim, D.E., Kuruoglu, R., Bradley, R.J., Dwyer, D., 1992. Diagnostic sensitivity of the laboratory tests in myasthenia gravis. Muscle Nerve 15, 720–724. Poulas, K., Koutsouraki, E., Kordas, G., Kokla, A., Tzartos, S.J., 2012. Anti-MuSK- and antiAChR-positive myasthenia gravis induced by D-penicillamine. J. Neuroimmunol. 250, 94–98. Rajakulendran, S., Viegas, S., Spillane, J., Howard, R.S., 2012. Clinically biphasic myasthenia gravis with both AChR and MuSK antibodies. J. Neurol. 259, 2736–2739. Sanders, D.B., Juel, V.C., 2008. MuSK-antibody positive myasthenia gravis. Questions from the clinic. J. Neuroimmunol. 15, 85–89. Saulat, B., Maertens, P., Hamilton, W.J., Bassam, B.A., 2007. Anti-musk antibody after thymectomy in a previously seropositive myasthenic child. Neurology 69, 803–804. Stickler, D.E., Massey, J.M., Sanders, D.B., 2005. MuSK-antibody positive myasthenia gravis: clinical and electrodiagnostic patterns. Clin. Neurophysiol. 116, 2065–2068. Suhail, H., Vivekanandhan, S., Sing, S., Behari, M., 2010. Coexistent of muscle specific tyrosine kinase and acetylcholine receptor antibodies in a myasthenia gravis patient. Neurol. India 58, 668–669. Trakas, N., Zisimopoulou, P., Tzartos, S.J., 2011. Development of a highly sensitive diagnostic assay for muscle specific tyrosine kinase (MuSK) autoantibodies in myasthenia gravis. J. Neuroimmunol. 15, 79–86. Vincent, A., Waters, P., Leite, M.I., Jacobson, L., Koneczny, I., Cossins, J., Beeson, D., 2012. Antibodies identified by cell-based assays in myasthenia gravis and associated diseases. Ann. N. Y. Acad. Sci. 1274, 92–98. Zouvelou, V., Kyriazi, S., Rentzos, M., Belimezi, M., Micheli, M.A., Tzartos, S.J., Stamboulis, E., 2013. Double-seropositive myasthenia gravis. Muscle Nerve 47, 465–466.
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
AChR-myasthenia gravis switching to double-seropositive several years after the onset Vasiliki Zouvelou a,⁎, Paraskevi Zisimopoulou b, Erasmia Psimenou c, Eirini Matsigkou b, Eleftherios Stamboulis a, Socrates J. Tzartos b,d a
Neurology Department, Aeginition Hospital, University of Athens, Greece Department of Biochemistry, Hellenic Pasteur Institute, Greece Renal Unit, Alexandra Hospital, Athens, Greece d Tzartos NeuroDiagnostics, Athens, Greece b c
a r t i c l e
i n f o
Article history: Received 29 September 2013 Received in revised form 8 December 2013 Accepted 17 December 2013 Keywords: Myasthenia gravis Acetylcholine receptor Muscle-specific kinase Double seropositive Cell-based assay Biphasic course
a b s t r a c t We report an early onset AChR-myasthenia gravis (MG) with biphasic clinical course. The clinical “switch” from AChR-MG to MuSK-MG emerged 16 years after the onset and 11 years after thymectomy. MuSK antibodies were detected only by cell-based assay and only upon clinical “switch”, while AChR antibodies remained positive and at high titers during the whole disease course. Although the occurrence of AChR antibodies and MuSK antibodies in the same individual is rare, the re-assessment of the antibody status, using all available assays, is advisable when there is clinical indication. © 2013 Elsevier B.V. All rights reserved.
1. Introduction
2. Case report
Antibodies to muscle-specific tyrosine kinase (MuSK Abs) are detected in approximately 40% of generalized acetylcholine receptor (AChR) antibody-negative myasthenia gravis (MG). MuSK-MG is a distinct clinical entity and its phenotype differs from AChR-MG. Occurrence of AChR antibodies (Abs) and MuSK Abs in the same individual is rare. The limited references concern double seropositivity detected at the onset of the disease (Zouvelou et al., 2013), years after the onset (Suhail et al., 2010) and “seroconversion” from AChR Abs to MuSK Abs taking place after thymectomy (Saulat et al., 2007; Sanders and Juel, 2008; Kostera-Pruszczyk and Kwiecinski, 2009). Routine testing for double seropositivity is not recommended in clinical practice, unless there is clinical indication. We report a female patient with AChR-MG which became double seropositive-MG many years after the onset. The reason for the re-assessment of antibody status was the biphasic clinical course which clearly indicated an immunological peculiarity.
A 36-year old Caucasian female with AChR-MG first came to our attention on January 2013. The onset of the disease was 17 years ago (1996), at the age of 19, when the presenting symptoms were diplopia and unilateral eyelid ptosis. Three months later MG generalized with upper and lower limbs involvement. Serum AChR Abs were positive at a high titer of 270 nM (positive ≥ 0.6 nM). She received only pyridostigmine for the first 5 years and in 2001 prednisolone was added to the regimen. On December 2001, she underwent thymectomy and the histological examination revealed follicular hyperplasia. In 2003 and in 2008 the patient gave birth to two offsprings suffering both from transient neonatal MG. At those periods the patient's titer of AChR Abs was 370 nM and 587 nM, respectively. From 1996 until 2012, the myasthenic weakness was restricted to ocular and limb muscles, well controlled with pyridostigmine and prednisolone and there was no episode of myasthenic crisis or requirement for short term immunotherapy i.e. plasma exchange (PE) or intravenous immunoglobulin. On May 2012, 16 years after the onset of MG, an unprovoked and severe clinical deterioration supervened which was characterized by prominent bulbar, facial, neck and respiratory muscle involvement with dramatic response to PE. The above-mentioned muscles had never been previously involved during the disease course. The patient received high dose prednisolone and cyclosporine (200 mg/d) and she remained on monthly courses of PE until the end of November 2012. At our first evaluation (early January 2013) the patient was in minimal
⁎ Corresponding author at: Department of Neurology, University of Athens, 74 Vas. Sophias Ave., Athens 11528, Greece. Tel.: +30 2107289404; fax: +30 2107216474. E-mail address: [email protected] (V. Zouvelou). 0165-5728/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jneuroim.2013.12.012
112
V. Zouvelou et al. / Journal of Neuroimmunology 267 (2014) 111–112
manifestations, as she reported no symptoms and the neurological examination revealed only mild fatigable lower limbs. MRI of the mediastinum did not show any residual thymic tissue. We considered that the last reported pattern of myasthenic weakness was unusual for longstanding and previously stable AChR-MG and highly indicative of MuSK-MG. For that reason we asked for re-assessment of antibody status. AChR Abs were positive at a titer of 366 nM, i.e. similar to her previous titers, while testing for MuSK Abs by a commercial radioimmunoprecipitation assay (RSR Ltd, Cardiff) and by an in house radioimmunoprecipitation assay (Trakas et al., 2011) was negative. However, a live Cell Based Assay (CBA) for MuSK Abs was then performed (according to Leite et al., 2008). Interestingly, the recent serum was found anti-MuSK positive by the CBA (at both test dilutions: 1:10 and 1:20), whereas sera derived during 2008, were found negative even at 1:10 dilution. At the end of January 2013, the patient reported for the first time asymmetric distal weakness of the hand, especially of the finger extensors, interfering with daily activities. This new pattern of weakness was directly attributed to MG, based on the findings of appropriate electrophysiological testing. Prednisolone was increased up to 60 mg/d with mild improvement of the distal weakness. While she was on high-dose prednisolone an exacerbation came up with moderate to severe bulbar and respiratory symptoms which responded to courses of PE. However, the distal weakness remained unaffected to the PE and it was the prominent disabling symptom until the last evaluation on September 2013. 3. Discussion The clinical course of our patient until May 2012 was rather typical of early onset AChR-MG. Later on, the pattern of the disease was more typical of MuSK-MG, as evidenced by the distribution of myasthenic weakness and the unprovoked exacerbations. The phenotype of MuSK-MG appears to be clinically distinct with individuals generally exhibiting prominent oculobulbar, neck and respiratory muscle involvement. Furthermore, exacerbations are common and can occur in the absence of triggering factors and despite high dose immunosuppression (Guptill et al., 2011). The finger extensor weakness of our patient emerged during the last year, it greatly interferes with daily activities and is refractory to immunotherapy, even PE. Although weakness of distal extremity muscles is unusual in MG, the propensity for finger extensor weakness has been described in the limited references of distal weakness in MG (Nations et al., 1999). Whereas repetitive nerve stimulation of distal muscles is normal in many MG patients, single-fiber electromyography (SF-EMG) of the extensor digitorum communis (EDC) muscle reveals excessive jitter in the majority of generalized MG patients (Oh et al., 1992). This finding emphasizes that neuromuscular transmission is impaired in the finger extensors of many MG patients, even if there is no clinical weakness. Distal weakness is highly unusual finding in MuSK-MG. However, in some of these patients an abnormal jitter in the EDC muscle can be found on SF-EMG (Stickler et al., 2005). In light of the biphasic clinical course of our patient, serological reassessment was undertaken which revealed the presence of both AChR Abs and MuSK Abs. Interestingly, the titer of AChR Abs remained high during the whole course, despite the immunotherapy, the thymectomy and the radiological absence of residual thymic tissue. MuSK Abs were not detected by the classical radioimmunoprecipitation assay but only by the novel CBA assay, which is known to detect MuSK Abs in several previously MuSK Ab negative patients (Vincent et al., 2012). This may suggest that MuSK CBA should be applied in both AChR Ab positive and negative patients with MuSK phenotype. The occurrence of both AChR Abs and MuSK Abs in the same individual is rare. We have recently reported a double seropositive case at the onset of MG and prior to any therapeutic intervention. The patient was a 36-year old woman with oculobulbar distribution of myasthenic weakness, responder to pyridostigmine and prednisolone and with low titer
of AChRAbs (Zouvelou et al., 2013). A double-seropositive MG patient has also been identified 8 years after the onset of the disease and 6 years after thymectomy. The antibody status at the onset or prior to thymectomy was not known (Suhail et al., 2010). Another case of double-seropositive MG patient was identified after administration of D-penicillamine, where both antibodies disappeared after D-penicillamine discontinuation (Poulas et al., 2012). Furthermore, there are three reports of childhood onset AChR-MG who “seroconverted” to MuSK-MG after thymectomy, possibly as a result of “epitope spread”. None had MuSK Ab titers checked at the onset and all had relatively low AChR Abs (Saulat et al., 2007; Sanders and Juel, 2008; KosteraPruszczyk and Kwiecinski, 2009). Recently, a case of clinically biphasic MG with both AChR and MuSK Abs has been reported (Rajakulendran et al., 2012). The onset was at age 13 with bulbar predilection of the myasthenic weakness. The patient underwent thymectomy and responded favorably for over 15 years, when exacerbations started to occur. In contrast to our case, AChR or MuSK Abs had never been tested before clinical “switch” i.e. at the onset, before thymectomy or during the clinical stable state. We have documented that our patient was AChR-positive at the onset, prior to thymectomy and after thymectomy till the “switch” of the phenotypic pattern. Indeed, the double seropositivity was detected upon clinical “switch” from a predominantly AChR-MG to a MuSK-MG. We excluded the possibility of its previous existence as sera derived during 2008 were found negative for MuSK antibodies tested both by radioimmunoprecipitation assay and CBA. The development of MuSK Abs many years after the disease onset and thymectomy as well the persistence of high titers of AChR Abs is intriguing. A plausible explanation for the emergence of MuSK Abs would be the immunological changes following thymectomy in AChR-MG. In particular, these immunological changes consist in an expansion in the T cell repertoire and a greater prevalence of autoantibodies and autoimmune diseases than in non-thymectomy cases (Gerli et al., 1999).
References Gerli, R., Paganelli, R., Cossarizza, A., Muscat, C., Piccolo, G., Barbieri, D., Mariotti, S., Monti, D., Bistoni, O., Raiola, E., Venanzi, F.M., Bertotto, A., Franceschi, C., 1999. Long-term immunologic effects of thymectomy in patients with myasthenia gravis. J. Allergy Clin. Immunol. 103, 865–872. Guptill, J.T., Sanders, D.B., Evoli, A., 2011. Anti-MuSK antibody myasthenia gravis: clinical findings and response to treatment in two large cohorts. Muscle Nerve 44, 36–40. Kostera-Pruszczyk, A., Kwiecinski, H., 2009. Juvenile seropositive myasthenia gravis with anti-MuSK antibody after thymectomy. J. Neurol. 256, 1780–1781. Leite, M.I., Jacob, S., Viegas, S., Cossins, J., Clover, L., Morgan, B.P., Beeson, D., Willcox, N., Vincent, A., 2008. IgG1 antibodies to acetylcholine receptors in “seronegative” myasthenia gravis. Brain 131, 1940–1952. Nations, S.P., Wolfe, G.I., Amato, A.A., Jackson, C.E., Bryan, W.W., Barohn, R.J., 1999. Distal myasthenia gravis. Neurology 52, 632–634. Oh, S.J., Kim, D.E., Kuruoglu, R., Bradley, R.J., Dwyer, D., 1992. Diagnostic sensitivity of the laboratory tests in myasthenia gravis. Muscle Nerve 15, 720–724. Poulas, K., Koutsouraki, E., Kordas, G., Kokla, A., Tzartos, S.J., 2012. Anti-MuSK- and antiAChR-positive myasthenia gravis induced by D-penicillamine. J. Neuroimmunol. 250, 94–98. Rajakulendran, S., Viegas, S., Spillane, J., Howard, R.S., 2012. Clinically biphasic myasthenia gravis with both AChR and MuSK antibodies. J. Neurol. 259, 2736–2739. Sanders, D.B., Juel, V.C., 2008. MuSK-antibody positive myasthenia gravis. Questions from the clinic. J. Neuroimmunol. 15, 85–89. Saulat, B., Maertens, P., Hamilton, W.J., Bassam, B.A., 2007. Anti-musk antibody after thymectomy in a previously seropositive myasthenic child. Neurology 69, 803–804. Stickler, D.E., Massey, J.M., Sanders, D.B., 2005. MuSK-antibody positive myasthenia gravis: clinical and electrodiagnostic patterns. Clin. Neurophysiol. 116, 2065–2068. Suhail, H., Vivekanandhan, S., Sing, S., Behari, M., 2010. Coexistent of muscle specific tyrosine kinase and acetylcholine receptor antibodies in a myasthenia gravis patient. Neurol. India 58, 668–669. Trakas, N., Zisimopoulou, P., Tzartos, S.J., 2011. Development of a highly sensitive diagnostic assay for muscle specific tyrosine kinase (MuSK) autoantibodies in myasthenia gravis. J. Neuroimmunol. 15, 79–86. Vincent, A., Waters, P., Leite, M.I., Jacobson, L., Koneczny, I., Cossins, J., Beeson, D., 2012. Antibodies identified by cell-based assays in myasthenia gravis and associated diseases. Ann. N. Y. Acad. Sci. 1274, 92–98. Zouvelou, V., Kyriazi, S., Rentzos, M., Belimezi, M., Micheli, M.A., Tzartos, S.J., Stamboulis, E., 2013. Double-seropositive myasthenia gravis. Muscle Nerve 47, 465–466.