International Journal of Neuroscience, 2014; 124(7): 486–490 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 0020-7454 print / 1543-5245 online DOI: 10.3109/00207454.2013.858337
RESEARCH ARTICLE
Calcium channel antibodies in patients with absence epilepsy 1 ¨ ¨ 1 Betul ¨ Baykan,1 Esme Ekizo˘glu,1 Canan Ulusoy,2 Zeynep Aydin-Ozemir, Pınar Tekturk, 1 3 2 ˙ ¸ oz, ¨ ¨ un ¨ Sema Ic Demet Kınay, and Erdem Tuz 1
Department of Neurology and Clinical Neurophysiology, Istanbul Medical Faculty, Istanbul University, Capa, Istanbul, Turkey; 2 Department of Neuroscience, Experimental Medicine Research Institute, Istanbul University, Capa, Istanbul, Turkey; 3 Department of Neurology, Bakirkoy State Hospital, Istanbul, Turkey Autoimmunity has aroused interest in the last years as a contributory mechanism of epilepsy, especially in epilepsies with unknown cause or therapy resistance. Since the relationship of absence epilepsy (AE) with calcium channels is well established, we aimed to investigate related antibodies in patients diagnosed with AE. Consecutive patients with typical absence seizures having either childhood absence epilepsy (CAE) or juvenile absence epilepsy (JAE) with generalized spike and wave discharges on electroencephalography (EEG) were included after their consent. The patients were diagnosed according to the International League Against Epilepsy (ILAE) 2010 criteria. Antibodies against P-Q type voltage gated calcium channels (VGCC) and T-type VGCC subunit Cav3.2 (encoded by the CACNA1H gene) were investigated by RIA and ELISA, respectively. We searched for these antibodies in 32 patients with AE and 53 patients with focal epilepsy of unknown cause (FEOUC) as the disease control group; furthermore, 30 healthy persons served as the healthy controls. Eleven patients (34.3%) with AE had CAE and the remaining patients had JAE. Only a 47-year-old female FEOUC patient, who also had systemic lupus erythematosus with normal MRI scans showed antibodies against P-Q type VGCC, whereas no antibody positivity could be found in other FEOUC and AE patients and healthy controls. Our results might suggest that calcium channel antibodies do not play an important role in the pathophysiology of AE. Further studies with larger groups of other epileptic syndromes are needed to confirm our results. KEYWORDS: absence epilepsy, autoimmunity, channelopathy
Introduction Autoimmunity as a contributory mechanism of epilepsy has attracted increased attention in recent years. Several well-known antibodies associated with epilepsies are directed against ion channels, receptors and associated proteins such as voltage-gated potassium channel (VGKC), glutamic acid decarboxylase (GAD), N-methyl-D-aspartate receptor (NMDAR), γaminobutyric acid receptor (GABAR)B and α-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) [1–6]. Antibodies against voltage-gated calcium channels (VGCC) were reported in a few Received 12 September 2013; revised 20 October 2013; accepted 20 October 2013 ¨ un, ¨ Department of Neurology, Istanbul Medical Correspondence: Erdem Tuz Faculty, Istanbul University, Capa/Fatih Istanbul, Turkey. Tel. +90 212 414 20 00-32593; +90 532 564 30 23. Fax: +90 212 631 05 03. E-mail: [email protected]
486
studies and only one patient was found to harbor these antibodies [7,8]. Two forms of absence epilepsy (AE), childhood absence epilepsy (CAE) and juvenile absence epilepsy (JAE), are well-established epilepsy syndromes with typical clinical and EEG findings [9]. They were accepted as epileptic syndromes with a good prognosis and a genetic background but their entire genetic profiles could not be elucidated despite many efforts in most of the patients [10]. Furthermore, there are patients with AE displaying resistance to antiepileptic drugs or other unusual findings such as mental or psychiatric problems implying that the underlying mechanisms may not be uniform. P/Q-type calcium channels play an important role in regulating membrane excitability, whereas T-type calcium channels are involved in spike-wave rhythmicity of corticothalamic circuitry [11,12]. We have recently identified antibodies to T-type VGCC subunit Cav3.2 (encoded by the CACNA1H gene) in headache with neurological deficits and cerebrospinal fluid
Calcium channel antibodies and absence epilepsy
lymphocytosis (HaNDL) patients, suggesting that the T-type VGCC might potentially be an antigenic target in other neurological disorders [13]. Since the relationship of AE with calcium channels is well known and there are many studies supporting the relationship between autoantibodies and epilepsy, we aimed to investigate calcium channel antibodies in sera of AE patients and control groups.
487
from the protein-coated well was subtracted from the noncoated well. The obtained results were expressed as signal ratios (sample signal/mean signal of the healthy controls). Positivity was defined as two standard deviations above the mean of healthy controls. Sera of three Lambert-Eaton myasthenic syndrome (LEMS) patients and two HaNDL patients were used as positive controls for P/Q-type calcium channel and Cav3.2 antibodies, respectively. All patient and control sera were tested at the same time.
Methods Patients Consecutive patients diagnosed with typical absence seizures as having CAE or JAE syndromes according to their age at onset and having typical generalized spike and wave discharges on their electroencephalography (EEG) were included in the study group after their signed consents. This study was approved by the local ethics committee. Patients with focal epilepsy of unknown cause (FEOUC) and also healthy subjects comprised the two control groups. All patients were diagnosed according to the 2010 International League Against Epilepsy (ILAE) criteria [9]. Subjects participating in the study were investigated for possible autoimmune disorders and the occurrence of autoimmune disorder was not an exclusion criteria. Only one patient in the FEOUC cohort had systemic lupus erythematosus (SLE), whereas none of other patients and controls had a concomitant autoimmune disease. The related clinical, EEG and neuroimaging findings were investigated from their files for the syndrome diagnosis.
Calcium channel antibody detection P/Q-type calcium channel antibody levels were measured by an RIA kit, as per manufacturer’s instructions and serum levels over 30 pM were accepted as positive (RSR Ltd., Cardiff, UK). Cav3.2 antibody levels were measured by ELISA as described previously [13]. The purified recombinant human Cav3.2 protein (1 μg/ml) was added to the wells of a 96-well plate and incubated overnight at 4◦ C. Noncoated wells were used as controls. Each serum sample was added in TBS-T (1:100) and incubated overnight at 4◦ C. The plates were then incubated with alkaline phosphatase (AP)-conjugated goat antihuman IgG (1:2000) (Southern Biotech, Birmingham, AL, USA) at room temperature for 1 h. After washing, 2-(2-benzothiazoyl)-6hyroxybenzothiazole phosphate (BBTP) was added for 45 min at room temperature followed by addition of the stopping solution (3N NaOH). Fluorescent signals were measured at 450/50 exitation and 580/50 emission with a microplate reader. For each sample, the value obtained C
2014 Informa Healthcare USA, Inc.
Statistical analysis Serum antibody levels were compared among AE, FEOUC and healthy control groups by ANOVA and Tukey’s post-hoc test. A p value of
RESEARCH ARTICLE
Calcium channel antibodies in patients with absence epilepsy 1 ¨ ¨ 1 Betul ¨ Baykan,1 Esme Ekizo˘glu,1 Canan Ulusoy,2 Zeynep Aydin-Ozemir, Pınar Tekturk, 1 3 2 ˙ ¸ oz, ¨ ¨ un ¨ Sema Ic Demet Kınay, and Erdem Tuz 1
Department of Neurology and Clinical Neurophysiology, Istanbul Medical Faculty, Istanbul University, Capa, Istanbul, Turkey; 2 Department of Neuroscience, Experimental Medicine Research Institute, Istanbul University, Capa, Istanbul, Turkey; 3 Department of Neurology, Bakirkoy State Hospital, Istanbul, Turkey Autoimmunity has aroused interest in the last years as a contributory mechanism of epilepsy, especially in epilepsies with unknown cause or therapy resistance. Since the relationship of absence epilepsy (AE) with calcium channels is well established, we aimed to investigate related antibodies in patients diagnosed with AE. Consecutive patients with typical absence seizures having either childhood absence epilepsy (CAE) or juvenile absence epilepsy (JAE) with generalized spike and wave discharges on electroencephalography (EEG) were included after their consent. The patients were diagnosed according to the International League Against Epilepsy (ILAE) 2010 criteria. Antibodies against P-Q type voltage gated calcium channels (VGCC) and T-type VGCC subunit Cav3.2 (encoded by the CACNA1H gene) were investigated by RIA and ELISA, respectively. We searched for these antibodies in 32 patients with AE and 53 patients with focal epilepsy of unknown cause (FEOUC) as the disease control group; furthermore, 30 healthy persons served as the healthy controls. Eleven patients (34.3%) with AE had CAE and the remaining patients had JAE. Only a 47-year-old female FEOUC patient, who also had systemic lupus erythematosus with normal MRI scans showed antibodies against P-Q type VGCC, whereas no antibody positivity could be found in other FEOUC and AE patients and healthy controls. Our results might suggest that calcium channel antibodies do not play an important role in the pathophysiology of AE. Further studies with larger groups of other epileptic syndromes are needed to confirm our results. KEYWORDS: absence epilepsy, autoimmunity, channelopathy
Introduction Autoimmunity as a contributory mechanism of epilepsy has attracted increased attention in recent years. Several well-known antibodies associated with epilepsies are directed against ion channels, receptors and associated proteins such as voltage-gated potassium channel (VGKC), glutamic acid decarboxylase (GAD), N-methyl-D-aspartate receptor (NMDAR), γaminobutyric acid receptor (GABAR)B and α-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) [1–6]. Antibodies against voltage-gated calcium channels (VGCC) were reported in a few Received 12 September 2013; revised 20 October 2013; accepted 20 October 2013 ¨ un, ¨ Department of Neurology, Istanbul Medical Correspondence: Erdem Tuz Faculty, Istanbul University, Capa/Fatih Istanbul, Turkey. Tel. +90 212 414 20 00-32593; +90 532 564 30 23. Fax: +90 212 631 05 03. E-mail: [email protected]
486
studies and only one patient was found to harbor these antibodies [7,8]. Two forms of absence epilepsy (AE), childhood absence epilepsy (CAE) and juvenile absence epilepsy (JAE), are well-established epilepsy syndromes with typical clinical and EEG findings [9]. They were accepted as epileptic syndromes with a good prognosis and a genetic background but their entire genetic profiles could not be elucidated despite many efforts in most of the patients [10]. Furthermore, there are patients with AE displaying resistance to antiepileptic drugs or other unusual findings such as mental or psychiatric problems implying that the underlying mechanisms may not be uniform. P/Q-type calcium channels play an important role in regulating membrane excitability, whereas T-type calcium channels are involved in spike-wave rhythmicity of corticothalamic circuitry [11,12]. We have recently identified antibodies to T-type VGCC subunit Cav3.2 (encoded by the CACNA1H gene) in headache with neurological deficits and cerebrospinal fluid
Calcium channel antibodies and absence epilepsy
lymphocytosis (HaNDL) patients, suggesting that the T-type VGCC might potentially be an antigenic target in other neurological disorders [13]. Since the relationship of AE with calcium channels is well known and there are many studies supporting the relationship between autoantibodies and epilepsy, we aimed to investigate calcium channel antibodies in sera of AE patients and control groups.
487
from the protein-coated well was subtracted from the noncoated well. The obtained results were expressed as signal ratios (sample signal/mean signal of the healthy controls). Positivity was defined as two standard deviations above the mean of healthy controls. Sera of three Lambert-Eaton myasthenic syndrome (LEMS) patients and two HaNDL patients were used as positive controls for P/Q-type calcium channel and Cav3.2 antibodies, respectively. All patient and control sera were tested at the same time.
Methods Patients Consecutive patients diagnosed with typical absence seizures as having CAE or JAE syndromes according to their age at onset and having typical generalized spike and wave discharges on their electroencephalography (EEG) were included in the study group after their signed consents. This study was approved by the local ethics committee. Patients with focal epilepsy of unknown cause (FEOUC) and also healthy subjects comprised the two control groups. All patients were diagnosed according to the 2010 International League Against Epilepsy (ILAE) criteria [9]. Subjects participating in the study were investigated for possible autoimmune disorders and the occurrence of autoimmune disorder was not an exclusion criteria. Only one patient in the FEOUC cohort had systemic lupus erythematosus (SLE), whereas none of other patients and controls had a concomitant autoimmune disease. The related clinical, EEG and neuroimaging findings were investigated from their files for the syndrome diagnosis.
Calcium channel antibody detection P/Q-type calcium channel antibody levels were measured by an RIA kit, as per manufacturer’s instructions and serum levels over 30 pM were accepted as positive (RSR Ltd., Cardiff, UK). Cav3.2 antibody levels were measured by ELISA as described previously [13]. The purified recombinant human Cav3.2 protein (1 μg/ml) was added to the wells of a 96-well plate and incubated overnight at 4◦ C. Noncoated wells were used as controls. Each serum sample was added in TBS-T (1:100) and incubated overnight at 4◦ C. The plates were then incubated with alkaline phosphatase (AP)-conjugated goat antihuman IgG (1:2000) (Southern Biotech, Birmingham, AL, USA) at room temperature for 1 h. After washing, 2-(2-benzothiazoyl)-6hyroxybenzothiazole phosphate (BBTP) was added for 45 min at room temperature followed by addition of the stopping solution (3N NaOH). Fluorescent signals were measured at 450/50 exitation and 580/50 emission with a microplate reader. For each sample, the value obtained C
2014 Informa Healthcare USA, Inc.
Statistical analysis Serum antibody levels were compared among AE, FEOUC and healthy control groups by ANOVA and Tukey’s post-hoc test. A p value of
