With the exception of rare Mendelian seizure disorders that are explained by point mutations in the genes encoding voltage- and ligand-gated ion channels (Rees, 2010), the molecular etiology of most epilepsies remains unclear. We and others have postulated that an autoimmune attack on protein components of the central nervous system (CNS) could underlie some epileptic disorders of currently unexplained causation (Palace & Lang, 2000; Kullmann, 2010; Irani et al., 2011a,b).
There are now several recognized neurologic disorders associated with serum antibodies to neurologic proteins, many of which present with seizures. Antibodies to the voltage-gated potassium channel (VGKC)-complex have been identified in patients with limbic encephalitis (LE; Thieben et al., 2004; Vincent et al., 2004; Irani et al., 2008, 2010a), and more recently in patients with faciobrachial dystonic seizures (FBDS; Irani et al., 2011a,b), antecedent to the onset of amnesia and disorientation. These encephalopathies often display a monophasic course with antibody titers that decline substantially over 1–2 years (Buckley et al., 2001). Patients typically respond poorly to treatment with conventional antiepileptic drugs (AEDs), often experiencing heightened adverse effects, but they respond well to immunotherapies (Irani et al., 2011a,b).
Serum antibodies to neurologic proteins are relatively common in patients presenting with acute seizures of suspected autoimmune origin, as determined by inflammatory changes in cerebrospinal fluid or on neuroimaging (Quek et al., 2012). Antibodies to VGKC complex proteins and to glutamic acid decarboxylase (GAD) have also been reported in a small proportion of patients presenting with seizures as the main or sole symptom and without overt autoimmune involvement (Kwan et al., 2000; McKnight et al., 2005; Majoie et al., 2006; Niehusmann et al., 2009). The majority of such reports have been cross-sectional in nature, often recruiting patients from tertiary referral centers and thereby biasing the study population in favor of those with an extended history of predominantly refractory epilepsy. Therefore, it remains unclear whether the elevated antibody titers observed in these studies are the underlying cause of the epilepsy or simply a consequence of uncontrolled seizures and any associated neurologic damage. Recently, antibodies to other brain-expressed proteins, including N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA), γ-aminobutyric acid receptor B (GABAB), and glycine receptors (GLY-Rs), have been additionally reported in patients with encephalopathies (Graus et al., 2010; Bien & Scheffer, 2011; Vincent & Crino, 2011), but the prevalence of these novel antibodies remains relatively unknown in larger cohorts of sporadic epilepsy. Whether they coexist with more widely reported antibodies, such as VGKC and GAD, in a seizure disorder with broad autoimmunity is equally unclear.
The aim of this study was to determine the prevalence of various established and novel autoantibodies in two large cohorts of people with epilepsy and to compare that prevalence in terms of demographics, clinical characteristics, and treatment outcome.
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A total of 416 people with epilepsy (CC = 235, NDC = 181) plus 148 controls (30 healthy volunteers, 98 neurologic controls, 20 autoimmune controls) were each tested for serum antibodies to VGKC, VGCC, GAD, NMDA-R, and GLY-R. A comparison of the CC and NDC patients is illustrated in Table 1. There were significantly more male patients in the NDC compared to the CC (p = 0.013). Age at enrollment into the study was similar between the two cohorts, but age at first-ever seizure was significantly higher in the NDC (p < 0.001), and significantly more patients in the NDC had unclassified epilepsy compared to focal epilepsy (p < 0.001). This is to be expected in a population of patients given a very recent diagnosis of epilepsy in an adult epileptology service.
Table 1. Characteristics of the epilepsy cohorts
| ||CC (n = 235)||NDC (n = 181)||p-Value|
|Median age at first seizure, years (range)||18 (0–83)||30 (3–82)||<0.001|
|Median age at study enrollment, years (range)||42 (16–83)||32.5 (16–82)||ns|
|Epilepsy type at enrollment|| || || |
A total of 46 epilepsy patients (CC and NDC combined) had serum antibodies to one or more of VGKC, NMDA-R, GAD, or GLY-R, a prevalence that was significantly (p < 0.001) higher than in the combined control cohort (Table 2). Full details of the antibody-positive patients are given in supplementary Table S1 (Table S1a NDC, Table S1b CC). The most common antibody was to the VGKC-complex, found in 20 epilepsy patients (4.8% of total; titer range 110–500 pm) and only one of the controls (0.7% of total), a stroke patient with a titer of 108 pm (Fig. 1; Table 2). Antibodies were also detected against GLY-R (2.6% of epilepsy patients), NMDA-R (1.7% of epilepsy patients), and GAD (1.7% of epilepsy patients), none of which were observed in the control cohort (Fig. 1; Table 2). One individual from CC had elevated titers to two different antibodies (VGKC and GLY-R) but without remarkable clinical features. A significant difference (p = 0.0271, Fisher's exact test) was observed in the prevalence of GLY-R antibodies between the two cohorts, this was lost after correction for multiple testing. There were no further significant differences in the relative prevalence of any of the other positive antibody titers, either collectively or individually, between CC and NDC patients (Table 2).
Table 2. Presence of neurologic autoantibodies in cases and controls
|Cases|| || || || || || || || || |
|Controlsa|| || || || || || || || || |
Figure 1. Patients with epilepsy (n = 416), disease controls (DC; n = 118), and healthy controls (HC; n = 30) were tested for autoantibodies to a range of neuronal proteins. (A) Scatter diagram showing results of screening for antibodies to voltage-gated potassium channels (VGKCs). Antibodies were measured by immunoprecipitation of 125I-α-dendrotoxin-labeled VGKCs (pm), with titers considered positive if >100 pm (calculated as three standard deviations above the mean observed in healthy controls). (B) Scatter diagram showing results of screening for antibodies to the NMDA receptor (NMDA-R). Antibodies were measured by cell-based assay, scored by two independent observers employing a semiquantitative scoring system ranging from 0 to 4, and considered positive if scoring an average of >1. (C) Scatter diagram showing results of screening for antibodies to the glycine receptor (GLY-R). Antibodies were measured by cell-based assay, scored by two independent observers employing a semiquantitative scoring system ranging from 0 to 4, and considered positive if scoring an average ≥2.
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None of the epilepsy patients (n = 416) or controls (n = 148) expressed antibodies to VGCC (Table 2). A random selection of CC and NDC patients were additionally investigated for the presence of antibodies to AQP4 (n = 90) and/or α7-AChR (n = 50), principally for the purposes of assay development and validation. None of these analyses was positive (data not shown).
All CC and NDC patients who had positive titers for VGKC (n = 20) plus 74 VGKC-negative patients (selected at random from CC and NDC) and 30 healthy controls were additionally tested for antibodies to the VGKC-complex associated proteins LGI1 and CASPR2 by CBA. Only one patient, with a positive VGKC-complex titer of 111 pm, had LGI1 antibodies. All other patients and controls were negative for LGI1 and all tests for CASPR2 were similarly negative. The LGI1 positive patient was a 61-year-old man with a 2-year history of focal dyscognitive seizures with oral automatisms (lip smacking) lasting a small number of minutes. He was subsequently retested and found to be negative for VGKC-complex and LGI1 antibodies following 4 months of treatment with controlled-release carbamazepine 400 mg twice daily.
There was no association between the presence of neurologic autoantibodies and age at enrollment and no difference in the relative prevalence of positive titers between males and females, when the CC and NDC cohorts were combined (Tables 3 and 4). Likewise, there was no difference in the presence of antibodies between patients with generalized, focal, or unclassified epilepsy, although all seven patients with elevated GAD antibodies (>100 units/ml) had focal epilepsy (Table 3). Further analysis of patients with focal epilepsy revealed a significantly higher prevalence of positive antibody titers in patients with focal epilepsies of unknown cause compared to patients with known structural/metabolic focal epilepsy (p < 0.02; Table 3). The duration of epilepsy at enrollment in the CC was not associated with the prevalence of elevated antibody titers, nor was the time since most recent seizure at enrollment in the NDC (p = ns, Table 4).
Table 3. Prevalence of neurologic autoantibodies by categorical demographic and clinical characteristics
|Sex|| || || || || || || || || |
|Epilepsy type|| || || || || || || || || |
|Etiologya|| || || || || || || || || |
Table 4. Prevalence of neurologic autoantibodies by continuous demographic and clinical characteristics
|Antibody status||Median age at enrollment in years (range; CC + NDC)||Median duration of epilepsy in years (range; CC only)||Median time since most recent seizure in days (range; NDC only)|
|Positive||42.00 (16–77)||18.2 (0.1–57)||19.8 (1–90)|
|Negative||40.78 (16–83)||13.77 (0.6–62)||20.3 (0–114)|
The final analysis explored antibody status at diagnosis and its potential relationship with subsequent response to AED therapy. It was performed solely in the NDC who were participating in a prospective, randomized trial of AED monotherapy, with patients designated as responders or nonresponders to treatment as described above. Individuals with an elevated antibody titer at baseline (irrespective of antibody) were more than twice as likely (16.4% vs. 7.9%) to be unresponsive to initial drug treatment, when measured at 6 months after AED initiation, than those who were antibody negative (Table 5). This observation, however, failed to reach statistical significance (p = 0.18). There were no significant differences in seizure-free rates at 6 months amongst the three AEDs under investigation in the monotherapy trial (levetiracetam, lamotrigine, topiramate; data not shown), ruling out any potentially confounding influence of treatment arm.
Table 5. Presence of neurological autoantibodies and response to first ever antiepileptic drug in newly-diagnosed cohort
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Previous studies have shown the presence of autoantibodies to VGKC complexes and GAD in cross-sectional cohorts of patients with epilepsy (Kwan et al., 2000; McKnight et al., 2005; Errichiello et al., 2009; Liimatainen et al., 2010; Irani et al., 2011a,b), in well-defined epilepsy syndromes (Irani et al., 2011a,b), and in patients with known or suspected preexisting neurologic autoimmunity (Quek et al., 2012). Antibodies to VGCC and GLY-R have never, to our knowledge, been systematically screened in the epilepsy population, and those to NMDA-R have only been investigated in two small pediatric studies, focused on LE and status epilepticus (Haberlandt et al., 2011; Suleiman et al., 2011). We looked for VGKC, VGCC, GAD, NMDA-R, and GLY-R antibodies in cohorts of consecutive, unselected patients with established epilepsy attending specialist clinics in Oxford and Sheffield and with newly diagnosed epilepsy taking part in a monotherapy trial in Glasgow.
We found positive antibodies to VGKC, GAD, NMDA-R, and GLY-R at a prevalence that was significantly higher than in either healthy or disease controls. In total, 46 (11%) of 416 patients with epilepsy in this analysis were antibody positive, with one patient showing an elevated titer to multiple antigens (VGKC and GLY-R). Prevalence of positive titers was comparable in patients with established and newly diagnosed epilepsy. There was no association with age, sex, epilepsy type, duration of epilepsy, or time since most recent seizure. There was a modest relationship with focal epilepsies of unknown cause and a trend toward AED unresponsiveness in patients with elevated antibody titers at diagnosis.
None of the patients in this study had clinical evidence of LE, NMDA-R–associated encephalitis, or any other antibody-mediated neuroinflammatory disease. As such, our findings suggest that the various neurologic autoantibodies associated with subacute onset encephalopathies may also give rise to sporadic epilepsy, similar to previous reports in a cohort of young female patients with unexplained new-onset epilepsy (Niehusmann et al., 2009). All seven patients with GAD antibodies had focal epilepsy, whereas six were female, which is consistent with a number of other studies (Kwan et al., 2000; Irani et al., 2011a,b) and more recent reports of a relationship between GAD antibodies and epilepsies arising in the temporal lobe (Peltola et al., 2000; Errichiello et al., 2009; Liimatainen et al., 2010; Irani et al., 2011a,b). Similarly, GAD antibodies have also recently been reported in a form of LE in young adult females with temporal lobe epilepsy and mild cognitive involvement (Malter et al., 2010). Five (8.6%) of the 58 patients with a diagnosis of generalized epilepsy had autoantibodies (three VGKC, two GLY-R), which is consistent with the relative prevalence of antibodies across our study as a whole. Patients with generalized epilepsies are usually presumed to have a genetic basis for their seizures, although in most cases this has not been formally confirmed. This issue is addressed in the recent revision to the terminology and concepts for organization of seizures and epilepsies (Berg et al., 2010). Autoantibodies directed against the same ion channels and receptors often implicated in genetic epilepsies could theoretically give rise to a clinically identical phenotype. Further studies on larger cohorts of generalized epilepsy patients to explore this possibility are thus warranted, and future revisions to the classification of epilepsies should consider autoimmunity as a significant etiologic contributor.
Uniquely, we tested 181 patients with a new diagnosis of epilepsy prior to the onset of treatment with AEDs and found a trend between antibody positivity and subsequent lack of response to the first-ever AED treatment. This is a preliminary finding and one that merits longer term follow-up in terms of drug responsiveness and further investigation to rule out potentially confounding influences on outcome. For example, it is possible that antibody-negative patients may have had a less acute and severe presentation, with lower pretreatment seizure number or density and an accordingly better prognosis. Nevertheless, this apparent lack of response to AEDs has been noted previously in VGKC-complex antibody-positive patients (Errichiello et al., 2009; Irani et al., 2011a,b) and there are increasing reports of VGKC antibody–positive patients who respond better to immunomodulatory therapy than to conventional AEDs (Lancaster et al., 2011; Quek et al., 2012). In a recent report of patients presenting with focal seizures and in whom an autoimmune etiology was suspected, 80% responded favorably to immunomodulation (Quek et al., 2012). Likewise in FBDS, a clinically distinctive syndrome associated with autoantibodies against LGI1 (Irani et al., 2010a) in which patients present with frequent brief, multiple episodes of facial and ipsilateral arm dystonia, electroencephalography (EEG) evidence of seizures, and progression to LE (Irani et al., 2008; Errichiello et al., 2009), few patients respond to AEDs, whereas immunotherapies have shown benefit (Irani et al., 2008; Errichiello et al., 2009).
Although the pathogenicity of the antibodies reported here has not been formally demonstrated, VGKC, GAD, NMDA-R and GLY-R antibody positive sera or immunoglobulin G (IgG) preparations thereof have all been shown to exert functional effects on neuronal tissues (Irani et al., 2010a,b; Lalic et al., 2011). As such, there are good reasons to believe that the antibodies detected in this study have the potential to be clinically significant, particularly when present early in the course of the disorder. Future immunologic screening studies should seek to preferentially include prospective cohorts of patients with new or recent-onset epilepsy to further understand the contribution of autoantibodies to the pathophysiology of epilepsy, to promote prompt and accurate diagnosis, and to encourage the consideration of alternative treatment options, including the possible use of immunotherapies.