FULL-LENGTH ORIGINAL RESEARCH Investigation of neuronal autoantibodies in two different focal epilepsy syndromes *Esme Ekizoglu, †Erdem Tuzun, ‡Mark Woodhall, ‡Bethan Lang, ‡Leslie Jacobson, *Sema Icoz, *Nerses Bebek, *Candan Gurses, *Aysen Gokyigit, ‡Patrick Waters, ‡Angela Vincent, and *Betul Baykan Epilepsia, 55(3):414–422, 2014 doi: 10.1111/epi.12528 SUMMARY Dr Ekizoglu has received her neurology training in UEMS Board certified, Istanbul Medical School. Objective: Neuronal antibodies have been identified in patients with seizures as the main or sole symptom. Our aim was to investigate the prevalence of these autoantibodies in patients with focal epilepsy of unknown cause (FEoUC) and in the group having mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS). Methods: We studied anti-neuronal antibodies of consecutive adult patients diagnosed with FEoUC and MTLE-HS in our epilepsy center. The clinical and laboratory features of antibody-positive patients were compared with those of seronegative patients. The responses to therapy have also been investigated. Results: Sera from 81 patients with epilepsy were tested. We found antibodies against glycine receptor (GLY-R) in 5 (6.2%), contactin-associated protein 2 (CASPR-2) in 4 (4.9%), N-methyl-D-aspartate receptor (NMDA-R) in 2 (2.5%), and voltage-gated potassium channel (VGKC)-complex in 2 (2.5%) of our patients with epilepsy. Psychotic attacks and nonspecific magnetic resonance imaging (MRI) white matter changes (WMCs) showed significant associations in seropositive patients (p = 0.003 and p = 0.03, respectively). Poor drug-response rates and total seizure counts were also higher in the seropositive patients but without reaching statistical significance. Three seropositive patients with previous epilepsy surgery showed typical histopathologic results for MTLE-HS, but not inflammatory changes. Moreover, some patients harboring these antibodies partly benefited from immunotherapy. Significance: We detected neuronal antibodies in one sixth of patients with focal epilepsy, GLY-R antibodies being the leading one. Psychosis or nonspecific MRI WMCs were frequent in the seropositive group. Our results suggested that relevant antibodies should be screened for a treatment possibility in these groups. KEY WORDS: Epilepsy, Autoantibodies, Glycine receptor, Voltage-gated potassium channel, N-methyl-D-aspartate receptor. Epilepsy is a common neurologic disorder showing resistance to antiepileptic drugs (AEDs) in at least 25–30% of the cases, and its etiology is still unknown.1,2 On the other hand, there is growing evidence that autoimmunity might play a role in epilepsy. A variety of serum antibodies to specific neuronal proteins has recently been identified in ordinary patients with epilepsy. Other than the first reported typical cases with limbic encephalitis or encephalopathy, Accepted November 26, 2013; Early View publication Feburary 6, 2014. *Department of Neurology, Epilepsy Center (EPIMER) and Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey; †Department of Neuroscience, Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey; and ‡Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom Address correspondence to Esme Ekizoglu, Department of Neurology, Istanbul University, Epimer Epilepsy Center and Istanbul Faculty of Medicine, Millet cad Capa, 34390 Istanbul, Turkey. E-mail: esmeekizoglu@yahoo.com This study was presented in a preliminary form as a poster in the 30th International Epilepsy Congress dated 23–27 June, held in Montreal, Canada. Wiley Periodicals, Inc. © 2014 International League Against Epilepsy 414 415 Neuronal Autoantibodies in Focal Epilepsies the following antigenic targets have been shown in patients who present with seizures as the main or sole symptom in recent years: voltage-gated potassium channel (VGKC)complex,3–5 N-methyl-D-aspartate receptor (NMDA-R),3,6 glutamic acid decarboxylase (GAD),3–5,7 and glycine receptors (GLY-Rs).3 A recent study demonstrated that 11% of two large unselected epileptic cohorts of new onset and with chronic course had antibodies to one or more of VGKC, NMDA-R, GAD, or GLY-R.3 Furthermore, an association between the autoimmune etiology and AED resistance has been reported, and a potential benefit of immunotherapy in improving seizure control has been suggested in some patients with antibodies to VGKC or GAD.5,8 Some of the reported seropositive patients also had mesial temporal lobe involvement.8 The exact clinical associations of these autoantibodies in various epilepsy syndromes are currently not well-established, and the clinicians want to know how to predict those patients who possibly harbor autoantibodies. Our aim was to investigate the prevalence of these autoantibodies in consecutive patients diagnosed with established focal epilepsy of unknown cause (FEoUC) and also in the group having mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS), which is mostly an AED-resistant and clinically distinct constellation, showing some inflammatory findings.1,9 Furthermore, we aimed to disclose the epilepsy profile of the patients who are seropositive for one of these neuronal autoantibodies to guide the clinicians who are managing patients with established epilepsy. Methods Participants We included all consecutive adult patients diagnosed with FEoUC or MTLE-HS, followed by our epilepsy center for more than 1 year. Our epilepsy center accepts all kinds of epilepsy patients and is not specific for drug-refractory patients. As a result we also follow many patients with a relatively benign course. The study was approved by the ethics committee. Informed consent was obtained from all participants before blood sampling. Seizures and syndromes were diagnosed according to the revised terminology, and concepts for organization of seizures and epilepsies of the International League Against Epilepsy (ILAE) Commission on Classification and Terminology,1 and their auras were classified according to Report of the ILAE Task Force on Classification and Terminology.10 The patients with obvious provoking factors or an apparent remote origin, such as a brain malformation or tumor, trauma, central nervous system infection with magnetic resonance imaging (MRI) evidence, or generalized epilepsy were excluded. Clinical parameters such as current age, age at onset, sex, all neurologic findings, and complaints like move- ment disorders, autonomic disturbances, sleep disturbances, medical and family history, epilepsy duration, seizure frequency, semiology, total seizure count in the previous year, types of aura, medication at the time of serum sampling, electroencephalography (EEG), videoEEG, MRI, and other laboratory findings were collected from the files of the patients and by reexamining and querying them with a standard form systematically, at the day of serum sampling by one of the authors. All MRI studies were performed with 1.5 T scanners with thin coronal in addition to sagittal and axial planes including T1, T2, and fluid-attenuated inversion recovery (FLAIR) images to visualize mesial temporal regions optimally. All EEG and video-EEG monitoring records were reviewed retrospectively by the authors. AED response was defined as poor in case of experiencing more than one seizure per month (with the exception of isolated auras) despite reasonable trials of two or more AEDs. The patients with MTLE, fulfilling these criteria once, who had undergone epilepsy surgery afterwards, were also grouped in the “poor prognosis” category, despite being better after surgery. Some of our patients have also been evaluated by a psychiatrist, and formal neuropsychological tests were done when clinically needed during the course. Autoantibody testing Sera from all patients and from 30 healthy controls were kept at 80°C until assayed. NMDA-R; a-amino-3hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPA-R); leucine rich, glioma-inactivated 1 (LGI1); contactin-associated protein-like 2 (CASPR-2); and GLY-R antibodies were detected by binding to HEK293 cells transfected with plasmids containing the NR1/NR2 subunits of the NMDA-R, GluR1/GluR2 subunits of the AMPA-R, LGI1, CASPR-2, or GLY-R a1 subunit, respectively. Transfected cells were incubated with patients’ sera (1:20) and the appropriate Alexa Fluor secondary antibody, as described earlier.3,6,11 The binding was scored visually on a range from 0 (negative) to 4 (very strong) as in previous studies.11 Only scores greater than one were accepted as positive to avoid nonspecific low positivity. For VGKCcomplex antibodies, radioimmunoassay (RIA) using brain extracts labeled with 125I-dendrotoxin (normal value <100 pM) were used.11,12 GAD antibodies were measured by immunoprecipitation of 125I-recombinant GAD.13 Immunotherapy was offered for seropositive patients with poor response to AEDs after their informed consent. Serum samples were obtained in a single patient with GLY-R antibody having improvement in seizure frequency after immunotherapy, at different time points and tested for this antibody at serial dilutions ranging from 1/10 to 1/320. In addition, the EEG spikes were counted before and after immune treatment in this patient. Epilepsia, 55(3):414–422, 2014 doi: 10.1111/epi.12528 416 E. Ekizoglu et al. Statistical analysis Descriptive statistics were applied, and the two groups of patients with and without serum antibodies were compared with chi-square tests, Fisher’s exact test, and independent samples t-test, where appropriate. No adjustments for multiple comparisons were done in this study. SPSS 15 software (SPSS Inc, Chicago, IL, U.S.A.) was used and the significance level was set at p < 0.05. Results A total of 81 consecutive patients with epilepsy, 55 with FEoUC (mean age: 39.0  13.8 years), 26 with MTLE-HS (mean age  standard deviation: 32.6  9.4 years) and 30 healthy volunteers (mean age: 36.4  8.1 years) were included in this study. The investigated autoantibodies were present in 13 patients (7 female, 6 male; mean age 35.5  8.0 years) with a seropositivity level of 16%. Antibodies were detected against GLY-R in 5 (6.2%), CASPR-2 in 4 (4.9%), NMDA-R in 2 (2.5%), and VGKC-complex in 2 (2.5%) of our patients with epilepsy (Tables 1 and 2). None of the CASPR-2 antibody–positive patients had VGKC-complex antibodies and vice versa. The seropositivity level did not decrease (15.7%) after excluding 11 tested patients with previously known autoimmune conditions such as systemic lupus erythematosus (SLE) and thyroiditis (shown in Table 3), from both of the seropositive and seronegative groups. None of the healthy subjects had any of the investigated auto-antibodies. In FEoUC group, we found GLY-R antibodies in 4, NMDA-R antibodies at low levels in 2 patients, and VGKCcomplex antibodies at low levels in one patient, whereas in MTLE-HS group, 4 patients were positive for antibodies to CASPR-2, one had GLY-R antibodies, and one had VGKCcomplex antibodies at low levels. Clinical and laboratory features of antibody-positive patients are given in Tables 1 and 2. Neurologic examinations were normal in the seropositive group, with the exception of two patients, who had mild essential tremor. The clinical and laboratory features and some comorbidities of the patient groups with and without autoantibodies are shown in Table 3, comparatively. The prevalence of psychotic attacks and nonspecific MRI white matter changes (WMCs) were found to be significantly higher in seropositive patients (p = 0.003 and p = 0.03, respectively). History of febrile seizures was relatively more frequent in patients having autoantibodies, but this difference did not reach statistical significance (Table 3). Three seropositive patients had remote history of head trauma without MRI evidence (Table 1) versus seven patients in the seronegative group (p > 0.05). Poor AED response and total seizure counts were higher in the seropositive group but without statistical significance. Epilepsy surgery was accomplished before serum sampling in three seropositive patients (3, 41, and 76 months ago) and also in 7 seronegative patients (median: 67, range: 32– Epilepsia, 55(3):414–422, 2014 doi: 10.1111/epi.12528 124 months ago) with beneficial results. The remaining seronegative patient has undergone epilepsy surgery 7 months after antibody testing. AED-resistant seropositive patients Among the total seropositive group, seven patients (three with FEoUC and four with MTLE-HS) had poor response to AEDs (Tables 1 and 2). We initiated immunotherapy for two of these FEoUC patients with poor response to AEDs. The remaining seropositive patient 1 (NMDA-R antibody positive) did not accept pulse steroid or intravenous immunoglobulin (IVIg) therapy. Seropositive patient 3 (GLY-R antibody positive) had early onset of seizures with a worsening course in years (5–15 seizures per day for more than 10 years), did not benefit from many AEDs or from vagus nerve stimulation. He was experiencing daily seizures at the time of first serum sampling. Intravenous methylprednisolone (IVMP) (1 g daily for 5 days followed by oral prednisolone 32 mg/day tapered off in 4 weeks) also remained ineffective, but afterward his seizure frequency improved with IVIg (0.4 g/kg/ day given for consecutive 5 days); he had 0–2 seizures per month during the subsequent follow-up period of 3 months. In addition, his EEG also showed some improvement, with 49% decrease of spike counts after IVIg therapy. We analyzed several serum samples from this patient at different time points. Although GLY-R antibody titers were slightly decreased after steroid and IVIg therapies, the titers fluctuated between 1/120 and 1/180 (scores between 2 and 3) throughout the treatment period. In other words, we did not find a strong correlation between antibody titers and number of seizures during each treatment episode. Seropositive patient 12 (VGKC-complex antibody positive) did not benefit markedly from pulse steroid and IVIg therapies, except for being seizure free for 3 weeks. One female patient with both MTLE and SLE (patient 8, CASPR-2 antibody positive) also had various immunotherapies. Her seizures did not benefit from steroid therapy and cyclophosphamide given during her previous follow-ups before antibody testing, and she remained seizure free for only 6 weeks under rituximab. Although having benefited from epilepsy surgery in the first years, seropositive patients 6 and 11 both experienced late recurrences of rare seizures after 3 and 4 years. Patient 9 was still seizure free following epilepsy surgery for a follow-up period of 1 year. The neuropathologic examinations of this three seropositive MTLE-HS patients (Tables 1 and 2, patients 6, 9, 11) who had undergone amygdalohippocampectomy, did not show any inflammatory changes and were not different from the seronegative patients with epilepsy surgery. In one of them (GLY-R antibody positive) CA1 predominant neuronal cell loss and gliosis were reported, whereas the other two patients (both CASPR-2 antibody positive) had more diffuse neuronal cell loss, with preservation of CA2 region. Table 1. The clinical and laboratory features of the antibody-positive patients No Antibodya Age sex Epilepsy syndrome 1 NMDA-R, 2 43 M FEoUC 2 NMDA-R, 2 39 M 3 4 GLY-R, 4 GLY-R, 4 5 Family history Psychiatric disorder FebS in the sister FEoUC Explosive onset with unknown etiologyb Unremarkable Postictal paranoid psychotic attacks 9 years after the onset Depression 37 M 19 M FEoUC FEoUC Unremarkable Unremarkable Unremarkable Consanguinity, FebS in the brother GLY-R, 3 30 F FEoUC Consanguinity, epilepsy in three cousins 6 GLY-R, 3 33 F Unremarkable Depression 7 GLY-R, 2 33 F MTLE-HS operated FEoUC Penetrating head trauma,c migraine Asthma 8 CASPR-2, 3 38 F MTLE-HS Blunt head trauma,c migraine, hyperthyroidism Systemic lupus erythematosus Consanguinity, epilepsy and mental retardation in a cousin Consanguinity of the parents 9 CASPR-2, 2 35 F Unremarkable 10 CASPR-2, 3 40 F MTLE-HS operated MTLE-HSd 11 CASPR-2, 2 36 M 12 VGKC (201.4 pM)e 53 F 13 VGKC (138.6 pM)e 26 M MTLE-HS operated FEoUC MTLE-HS (bilateral) Memory or cognitive comorbidities MRI findings PET findings Nonspecific white matter changes – Attention disorder and secondary memory disorder Not present Diffuse cognitive dysfunction Right F subcortical small nonspecific signal change – Normal Normal – Left superior P hypometabolism Normal – None Mild attention disorder and secondary memory disorder Verbal memory defect, frontal dysfunction Not present Depression Not present Unremarkable Depression Migraine Unremarkable Unremarkable Frontal lobe and left hemispheric dysfunction Not present Unremarkable Unremarkable None Verbal memory defect Hashimoto thyroiditis, MGUS, sleep problems Blunt head traumac Epilepsy in her sister (BCS) None Consanguinity, epilepsy in the cousin Psychosis 8 years after the onset, suicidal ideation Attention difficulty, mild frontal dysfunction, hypergraphism Attention disorder and secondary memory disorder Unremarkable Depression Psychotic spells 10 years after the onset, borderline personality disorder Depression Compatible with left HS Normal Compatible with left HS and small white matter changes Compatible with left HS Compatible with right HS Nonspecific small white matter changes Compatible with left HS Left T hypometabolism – – Bilateral mesial Thypometabolism – – Nonspecific white matter changes BiT left > right hypometabolism Compatible with bilateral HS BiT right > left hypometabolism N, normal; M, male; F, female; FEoUC, focal epilepsy of unknown cause; MTLE-HS, mesial temporal lobe epilepsy with hippocampal sclerosis; VGKC, voltage-gated potassium channel; CASPR-2, contactin-associated protein-like 2; NMDAR, N-methyl-D-aspartate receptor; GLY-R, glycine receptor; FebS, febrile seizure; MRI, magnetic resonance imaging; PET, positron emission tomography; Fe, ferritin; CBZ, carbamazepine; F, frontal; T, temporal; MGUS, monoclonal gammopathy of unknown significance; BCS, bilateral convulsive seizure. a Numbers indicate the antibody binding intensity scored visually on a range from 0 (negative) to 4 (very strong). b Lumbar puncture results normal (infectious etiologies and cancer were excluded). c Patient 5 had head trauma 5 years before the onset of seizures, patient 7 had trauma 8 years before the onset of seizures, patient 13 had trauma 4 years after the onset of seizures. d This patient has the typical MRI findings compatible with mesial temporal lobe sclerosis but her clinical picture is rather benign, as seen in the second part. e Healthy control <100 pM. 417 Epilepsia, 55(3):414–422, 2014 doi: 10.1111/epi.12528 Memory disorder Neuronal Autoantibodies in Focal Epilepsies History No Seizure types 1 FSwIoC, F-BCS 24 2 FebS, BCS 32 3 F-BCS 17 4 FSwIoC, F-BCS 3 5 FSwIoC, F-BCS 14 Cephalic, autonomic 6 FSwIoC, F-BCS 16 7 FSwIoC, F-BCS 16 8 FebS, FSwIoC, F-BCS 17 Epigastric, autonomic Auditory, affective Affective, autonomic 9 FebS, FSwIoC 21 Experiential, autonomic 10 11 FebS, FSwIoC FSwIoC, F-BCS 37 20 None Cephalic, affective 12 FSwIoC 46 None 13 FebS, FSwIoC, F-BCS 8 Types of aura Olfactory, autonomic, experiential Cephalic, autonomic None None Autonomic, affective Background EEG activity Interictal epileptic activity on routine EEG Ictal EEG (video-EEG monitoring) Other EEG findings AED at the sampling AED responsea Diffuse theta and delta waves to normal Left and right T rare sharp waves Not available – LEV 3500, TOP 300 Poor Left FT theta, otherwise normal Left FT theta, otherwise normal Diffuse theta Left FT spike Not available – VPA 1000 Good Left FT rare sharp waves Left CP >right CP spikes Not available – VPA 600, LEV 1500 Good Seven seizures starting in left F area (also subclinical seizures) Right FT subclinical seizures – CBZ 1200, LEV 2000, TOP 200, VNS Poor Right FIRDA CBZ 800 Good Three seizures starting in left FT area Not available Left TIRDA OXC 600, TOP 100 Poor – VPA 750 Good Fe deficiency anemia Not available Right FIRDA CBZ 1200, PRG 300 Poor Use of immunesuppressants Invasive recording 5 from left, two seizures from right FT area Not available Two seizures left FT (with switch off to right side) Five seizures starting in left FT area No seizures during 5 days of VEM – CBZ 1200, LEV 2000 Poor – Left TIRDA CBZ 400 CBZ 1200, LEV 1000, TOP 200 Good Poor – LEV 2000, ZON 200 Poor Right TIRDA LEV 3000 Good Paroxysmal slow waves, otherwise normal Left FT theta, otherwise normal Normal FT theta-delta paroxysms otherwise normal FT theta waves Right FT spikes Left FT spikes Right TP sharp waves and theta waves Right FT spikes Bi FT left > right spikes FT Theta waves Left FT theta-delta waves, otherwise normal BiFT L > R theta waves None Left FT sharp waves with phase reversal, Right T rare sharp waves Left > right FT spikes Right FT thetadelta waves, otherwise normal Right T sharp waves Other findings Prominent hyponatremia with CBZ Fever triggered seizures FSwIoC, focal seizure with impairment of consciousness; F-BCS, focal seizure evolving to bilateral convulsive seizure; FebS, febrile seizure; L, left; R, right; C, central; F, frontal; P, parietal; T, temporal; FIRDA, frontal interictal rhythmic delta activity; TIRDA, temporal interictal rhythmic delta activity; AED, antiepileptic drug; CBZ, carbamazepine; LEV, levetiracetam; PRG, pregabalin; TOP, topiramate; VPA, valproic acid; ZON, zonisamide; VNS, vagus nerve stimulation. a Good prognosis implied that the patient has less than one convulsive seizure of any kind monthly (auras excluded): Those patients who have undergone selective amygdalohippocampectomy were also classified in the poor prognosis group even if they reach remission after epilepsy surgery. 418 Age at onset E. Ekizoglu et al. Epilepsia, 55(3):414–422, 2014 doi: 10.1111/epi.12528 Table 2. The seizure and EEG characteristics of the antibody positive patients 419 Neuronal Autoantibodies in Focal Epilepsies Table 3. Comparison of the clinical features of patients with and without autoantibodies Sex (female/male) Age at serum sampling (years) Median; [Q3 Q1] Age at onset of epilepsy (years) Median; [Q3 Q1] Epilepsy duration (years) Median; [Q3 Q1] Total seizure count in the previous year Median; [Q3 Q1] Poor AED response (n) (%) History of status epilepticus (n) (%) Autonomic aura (n) (%) History of febrile seizures (n) (%) Coexisting autoimmune diseases (n) (%) Hashimoto’s thyroiditis Systemic lupus erythematosus (SLE) SLE and antiphospholipid syndrome Antiphospholipid syndrome Behcßet’s disease History of psychotic attacks (n) (%) Nonspecific MRI white matter changes (n) (%) Epilepsy surgery (n) (%) Seropositive patients (n = 13) Seronegative patients (n = 68) p-Values 6/7 36; [39.5–31.5] 28/40 36; [43.7–27.2] NS NS 17; [28–15] 23; [31.7–15.2] NS 16; [18.5–10.5] 12; [18–5.2] NS 18; [36–2.5] 2; [24–0] NS 7 (53.8) 0 (0) 7 (53.8) 5 (38.5) 2 (15.4) 1 1 – – – 3 (23.1) 5 (38.5) 3 (23.1) 31 (45.6) 3 (4.4) 22 (32.3) 13 (19.1) 9 (13.2) 4 1 2 1 1 0 (0) 8 (11.8) 8 (11.8) NS NS NS NS NS 0.003a 0.03a NS AED, antiepileptic drugs; MTLE-HS, mesial temporal lobe epilepsy with hippocampal sclerosis; MRI, magnetic resonance imaging; n, number; NS, not significant; Q1, first quartile; Q3, third quartile. Please note that interquartile range is IQR = Q3 Q1. a Fisher’s exact test two-sided. Discussion There is growing interest for possible autoimmune mechanisms in clinical epilepsy research, and even a concept of “autoimmune epilepsy “was recently proposed by different authors.8,14 We found that a remarkable number of patients with focal epilepsy with either unknown cause or MTLE-HS harbor various neuronal antibodies, GLY-R antibodies being the first (6.2%), followed by CASPR-2 (4.9%), NMDA-R (2.5%), and VGKC-complex (2.5%) antibodies. Our study was based on a consecutive series with established epilepsy from an epilepsy center that accepted patients with all kinds epilepsy, not specifically drug-refractory patients only. There are previous studies showing the presence of neuronal antibodies in some patients having epilepsy but with different inclusion criteria.3–5,8 In our series with focal epilepsy, we found autoantibodies in one sixth of the patients with epilepsy, with a higher rate than all previous reports, emphasizing the importance of a syndromic approach. Focal epilepsy of unknown cause and autoantibodies It was remarkable that GLY-R antibodies were detected in four patients in the FEoUC group. Antibodies directed to GLY-R were first reported in patients with progressive encephalomyelitis with rigidity and myoclonus.15,16 The intriguing association of GLY-R antibodies with epilepsy was reported for the first time in a recent study where GLYR antibodies were detected with 3% prevalence.3 Our study supported the importance of this antibody with a prevalence of 6.2%. Our cohort included syndromes of FEoUC and MTLE-HS, whereas the first study included all kinds of epilepsies, including genetic generalized forms. Because GLYR is a very recently discovered antibody, its exact incidence waits to be clarified. Future studies are needed to understand the contribution of GLY-R autoantibodies to the pathophysiology of epilepsy. VGKC-complex antibodies were reported previously in patients with FEoUC,3,4 and in association with drug-resistant focal epilepsy.5 Similarly, our VGKC-complex antibody-positive patient had a poor response to AEDs. In addition, we have detected NMDA-R antibodies in two patients having FEoUC but with different clinical pictures; one had explosive onset of seizures similar to limbic encephalitis (LE) with memory problems and poor response to AEDs followed by very late onset episodic postictal paranoid psychotic symptoms, whereas the second one had a mild course and did not show these features. Furthermore, these two patients did not show the reported extreme delta brush activity on careful analysis of all EEG studies.17 We suggest that clinicians should have a broader index of suspicion for NMDA-R antibody evaluation in patients with FEoUC, as this antibody might cause a wide clinical spectrum of disease. Epilepsia, 55(3):414–422, 2014 doi: 10.1111/epi.12528 420 E. Ekizoglu et al. MTLE-HS and autoantibodies Antibodies directed to the VGKC complex, GAD, and NMDA-R have already been related to LE associated with seizures.18–20 Furthermore the evolution from LE to TLE and to MTLE-HS, which cannot be distinguished from the MRI correlate of HS has been described.21–23 Our consecutive group of 26 MTLE-HS patients was larger than the previous studies where the investigators also found neuronal antibodies.4,5,8 Among our cases, four patients were positive for antibodies to CASPR-2 (but interestingly were negative for VGKC-complex antibodies assessed by RIA) and another one had VGKC-complex antibodies at low levels but no LGI1 or CASPR-2 antibodies. Hence 19.2% of our MTLE cases were shown to be associated with antibodies to the VGKC-complex, suggesting a potential role of VGKCcomplex autoimmunity in the pathogenesis of a subgroup of MTLE-HS patients. Furthermore another intriguing MTLE case had GLY-R antibody. To our knowledge both CASPR2 and GLY-R antibodies are reported for the first time in association with MTLE-HS syndrome. The implications of these findings remain to be elucidated in a larger group of patients with MTLE. Other investigated neuronal autoantibodies undetected in our series There have been some reports of GAD autoantibodies in patients with epilepsy.3,24 However, we could not detect any GAD antibody–positive case in this cohort. The frequency of GAD antibody in patients with epilepsy ranges from 0% to 7%.4,5,7,25 Therefore, the clinical role of GAD autoantibodies in epilepsy does not seem to be very clear currently.4,26 At the time of our study, we diagnosed a single case having GAD antibodies with nonconvulsive status epilepticus; she was not included because she was not admitted to the epilepsy center but was followed in the neurology ward and the details of this unique case are published elsewhere.27 Moreover, we could not detect any case with AMPA-R antibodies in our series, suggesting that this antibody does not play a pioneering role in epileptic seizures, confirming previous reports.3,4,28 We also did not detect any antibodies to LGI antigen.14 Coincidence of autoantibodies with other autoimmune disorders Some previous studies investigated autoantibodies in epileptic patients with coexisting autoimmune diseases.4,5 There is a well-known increased risk for patients with SLE to develop unprovoked seizures.29 The mechanism of this association is unsolved in patients without any obvious cause for seizures. Some antibodies such as antiphospholipid antibodies have been reported to be associated with seizures in SLE, but there are controversial results.5,30,31 Hashimoto’s thyroiditis (HT) is another frequent autoimmune disease, in which the association with epilepsy is not Epilepsia, 55(3):414–422, 2014 doi: 10.1111/epi.12528 very clear and the role of these antibodies in Hashimoto’s encephalopathy showing higher association with seizures is also uncertain.31History of other antibody-mediated disorders or organ-specific autoimmunity has been proposed as a supportive feature for neuronal antibody screening by Zuliani et al.32 We did not find an association between the presence of previously known autoimmune conditions and the seropositivity in our sample of consecutive patients, but two seropositive patients having coexisting autoimmune diseases had antibodies against potassium channels. Therefore, our findings may suggest that potassium channels could be another possible antigenic target in these patients with epilepsy. The clinical and laboratory associations of seropositive patients We compared the patients harboring neuronal antibodies with the seronegative patients and confirmed that the epilepsy duration did not seem to be important for the presence of autoantibodies as reported previously.3,4 The lack of association with epilepsy duration also partly contradicts the probability of the appearance of these antibodies as a result of neuronal destruction caused by chronic epilepsy. Our findings showed that WMCs on the MRI and psychotic spells showed significant associations with seropositivity, as seen in Table 3. It should be noted, however, that our group could be considered small and no statistical adjustments for multiple comparisons were done. Therefore, further studies with large series are needed to draw firm conclusions. Antibodies against NMDA-R were reported previously in new-onset extratemporal epilepsy with psychiatric symptoms.33 Two women in this report had WMCs on MRI, but this point was not further discussed. It is interesting to note that both of our patients with NMDA-R antibodies also had nonspecific WMCs. This radiologic finding was also seen in only one patient having established epilepsy with GLY-R antibodies.3 Furthermore, subcortical bifrontal and left parietal hyperintensities were reported in one 9-year-old encephalitis case with VGKC antibodies.23 The present study, however, extends previous anecdotal observations and suggests that WMCs seem to be a possible marker for the presence of autoantibodies in epilepsy patients and should also be included in the indication list for search of neuronal antibodies.33 This association may partly be related to the coexistence of patients with SLE, migraine, and HT, since all of these disorders are known to cause WMCs.31,34 It should be noted, however, that there are also patients with SLE, migraine, and HT without any autoantibodies in our series. These antibodies might cause direct immune-mediated changes in the brain ending up with WMCs, but it seems also highly likely, that they could be innocent bystanders. The association between anti-NMDA-R encephalitis and acute psychosis has been well documented.35–37 The presence of VGKC antibodies in psychotic patients was also 421 Neuronal Autoantibodies in Focal Epilepsies reported in a few patients.38,39 Therefore, the association of psychosis with neuronal antibodies in our epilepsy series is not a great surprise but indicates that patients with epilepsy and history of psychosis should be screened for these antibodies for a treatment possibility. On the other hand, one of our three cases with psychotic spells had GLY-R antibodies, adding this antibody to the list of antibodies associated with psychosis for the first time. Responses to treatment Poor AED response seemed higher in seropositive group without statistical significance. Other studies reported more patients with AED resistance, but they included epileptic patients with suspected autoimmune origin, which could have created a selection bias.8 There were antibody-positive patients with a benign clinical course in our series, thus immunotherapeutic interventions should be based on the individual’s AED response status. Two AED-resistant patients with antibodies against VGKC showed no clear benefit from immunotherapy trials despite having short-term seizure reductions for 3–6 weeks. We also assessed prospectively the response to immunotherapy in a patient having FEoUC with seropositivity for GLY-R antibody for the first time. IV pulse steroid treatment did not show any benefit, whereas IVIg treatment showed reduction of seizure frequency unrelated to antibody titers and moderate EEG response, but it is not possible to exclude the consequences of natural fluctuations and placebo effect. There is also a report of a GLY-R antibody positive case with an immunotherapy responsive isolated mesial temporal lobe status epilepticus.33 We are only aware of two neuronal antibody-positive patients, who have undergone epilepsy surgery without any benefit, in the relevant literature.8 Our three patients with MTLE-HS had undergone selective amydalohippocampectomy before antibody testing with favorable results and showed pathologic evidence of HS indistinguishable from the seronegative patients. Furthermore our results showed late relapses in two patients after successful epilepsy surgery. Moreover having previous epilepsy surgery also did not show any relation with antibody positivity excluding the appearance of antibodies in response to tissue destruction caused by surgery. In conclusion, there is a substantial number (one sixth of all) of patients with focal epilepsy with either unknown cause or MTLE-HS who harbor various neuronal antibodies, the recently reported GLY-R antibodies being the leading cause, followed by VGKC-complex antibodies in our series. We recommend in the light of our data that epileptic individuals with psychosis or nonspecific MRI WMCs be assessed with the possibility of antibody-mediated disorder in mind. The patients showing these serum antibodies could be treated with immunotherapy, when there is a clinical indication. There are still many unanswered questions such as the selection of the candidates for antibody testing, natural history of autoimmune epilepsy possible immunotherapeutic intervention types, and duration and the role of epilepsy surgery. In addition, these autoantibodies could be the markers of currently unknown immunopathologic processes rather than having a major role in pathogenesis. Future studies with larger series of epilepsy syndromes are needed to draw further conclusions. Disclosure None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines References 1. Berg AT, Berkovic SF, Brodie MJ, et al. Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005–2009. Epilepsia 2010;51:676–685. 2. Brodie MJ, Barry SJ, Bamagous GA, et al. Patterns of treatment response in newly diagnosed epilepsy. Neurology 2012;78:1548–1554. 3. Brenner T, Sills GJ, Hart Y, et al. Prevalence of neurologic autoantibodies in cohorts of patients with new and established epilepsy. Epilepsia 2013;54:1028–1035. 4. Majoie HJ, de Baets M, Renier W, et al. Antibodies to voltage-gated potassium and calcium channels in epilepsy. Epilepsy Res 2006;71:135–141. 5. McKnight K, Jiang Y, Hart Y, et al. Serum antibodies in epilepsy and seizure-associated disorders. Neurology 2005;65:1730–1736. 6. Irani SR, Bera K, Waters P, et al. N-methyl-D-aspartate antibody encephalitis: temporal progression of clinical and paraclinical observations in a predominantly non-paraneoplastic disorder of both sexes. Brain 2010;133:1655–1667. 7. Liimatainen S, Peltola M, Sabater L, et al. Clinical significance of glutamic acid decarboxylase antibodies in patients with epilepsy. Epilepsia 2010;51:760–767. 8. Quek AM, Britton JW, McKeon A, et al. Autoimmune epilepsy: clinical characteristics and response to immunotherapy. Arch Neurol 2012;69:582–593. 9. Yang T, Zhou D, Stefan H. Why mesial temporal lobe epilepsy with hippocampal sclerosis is progressive: uncontrolled inflammation drives disease progression? J Neurol Sci 2010;296:1–6. 10. Blume WT, Luders HO, Mizrahi E, et al. Glossary of descriptive terminology for ictal semiology: report of the ILAE task force on classification and terminology. Epilepsia 2001;42:1212–1218. 11. Irani SR, Alexander S, Waters P, et al. Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis. Morvan’s syndrome and acquired neuromyotonia. Brain 2010;133:2734–2748. 12. Motomura M, Johnston I, Lang B, et al. An improved diagnostic assay for Lambert-Eaton myasthenic syndrome. J Neurol Neurosurg Psychiatry 1995;58:85–87. 13. Vincent A, Grimaldi LM, Martino G, et al. Antibodies to 125Iglutamic acid decarboxylase in patients with stiff man syndrome. J Neurol Neurosurg Psychiatry 1997;62:395–397. 14. Irani SR, Bien CG, Lang B. Autoimmune epilepsies. Curr Opin Neurol 2011;24:146–153. 15. Hutchinson M, Waters P, McHugh J, et al. Progressive encephalomyelitis, rigidity, and myoclonus: a novel glycine receptor antibody. Neurology 2008;71:1291–1292. 16. Clerinx K, Breban T, Schrooten M, et al. Progressive encephalomyelitis with rigidity and myoclonus: resolution after thymectomy. Neurology 2011;76:303–304. 17. Schmitt SE, Pargeon K, Frechette ES, et al. Extreme delta brush: a unique EEG pattern in adults with anti-NMDA receptor encephalitis. Neurology 2012;79:1094–1100. Epilepsia, 55(3):414–422, 2014 doi: 10.1111/epi.12528 422 E. Ekizoglu et al. 18. Vincent A, Buckley C, Schott JM, et al. Potassium channel antibodyassociated encephalopathy: a potentially immunotherapy-responsive form of limbic encephalitis. Brain 2004;127:701–712. 19. Graus F, Saiz A, Lai M, et al. Neuronal surface antigen antibodies in limbic encephalitis: clinical-immunologic associations. Neurology 2008;71:930–936. 20. Haberlandt E, Bast T, Ebner A, et al. Limbic encephalitis in children and adolescents. Arch Dis Child 2001;96:186–191. 21. Bien CG, Elger CE. Limbic encephalitis: a cause of temporal lobe epilepsy with onset in adult life. Epilepsy Behav 2007;10:529–538. 22. Kroll-Seger J, Bien CG, Huppertz HJ. Non-paraneoplastic limbic encephalitis associated with antibodies to potassium channels leading to bilateral hippocampal sclerosis in a pre-pubertal girl. Epileptic Disord 2009;11:54–59. 23. Suleiman J, Brenner T, Gill D, et al. VGKC antibodies in pediatric encephalitis presenting with status epilepticus. Neurology 2011;76:1252–1255. 24. Aykutlu E, Baykan B, Gurses C, et al. No association of anti-GM1 and anti-GAD antibodies with juvenile myoclonic epilepsy: a pilot study. Seizure 2005;14:362–366. 25. Errichiello L, Perruolo G, Pascarella A, et al. Autoantibodies to glutamic acid decarboxylase (GAD) in focal and generalized epilepsy: a study on 233 patients. J Neuroimmunol 2009;211:120–123. 26. Saiz A, Blanco Y, Sabater L, et al. Spectrum of neurological syndromes associated with glutamic acid decarboxylase antibodies: diagnostic clues for this association. Brain 2008;131:2553–2563. 27. Cikrikcßili U, Ulusoy C, Turan S, et al. Non-convulsive status epilepticus associated with glutamic acid decarboxylase antibody. Clin EEG Neurosci 2013;44:232–236. 28. Lai M, Hughes EG, Peng X, et al. AMPA receptor antibodies in limbic encephalitis alter synaptic receptor location. Ann Neurol 2009;65:424– 434. Epilepsia, 55(3):414–422, 2014 doi: 10.1111/epi.12528 29. Adelow C, Andersson T, Ahlbom A, et al. Unprovoked seizures in multiple sclerosis and systemic lupus erythematosus: a populationbased case–control study. Epilepsy Res 2012;101:284–287. 30. Appenzeller S, Cendes F, Costallat LT. Epileptic seizures in systemic lupus erythematosus. Neurology 2004;63:1808–1812. 31. Vincent A, Crino PB. Systemic and neurologic autoimmune disorders associated with seizures or epilepsy. Epilepsia 2011;52(Suppl. 3):12– 17. 32. Zuliani L, Graus F, Giometto B, et al. Central nervous system neuronal surface antibody associated syndromes: review and guidelines for recognition. J Neurol Neurosurg Psychiatry 2012;83:638–645. 33. Niehusmann P, Dalmau J, Rudlowski C, et al. Diagnostic value of Nmethyl-D-aspartate receptor antibodies in women with new-onset epilepsy. Arch Neurol 2009;66:458–464. 34. Dinia L, Bonzano L, Albano B, et al. White matter lesions progression in migraine with aura: a clinical and MRI longitudinal study. J Neuroimaging 2013;23:47–52. 35. Barry H, Hardiman O, Healy DG, et al. Anti-NMDA receptor encephalitis: an important differential diagnosis in psychosis. Br J Psychiatry 2011;199:508–509. 36. Dalmau J, Gleichman AJ, Hughes EG, et al. Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies. Lancet Neurol 2008;7:1091–1098. 37. Lennox BR, Coles AJ, Vincent A. Antibody-mediated encephalitis: a treatable cause of schizophrenia. Br J Psychiatry 2012;200:92–94. 38. Parthasarathi UD, Harrower T, Tempest M, et al. Psychiatric presentation of voltage-gated potassium channel antibody-associated encephalopathy. Case report. Br J Psychiatry 2006;189:182–183. 39. Zandi MS, Irani SR, Lang B, et al. Disease-relevant autoantibodies in first episode schizophrenia. J Neurol 2011;258:686–688.