
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12765-X
10.1016/j.heliyon.2024.e36734
e36734
Research Article
N-methyl-D-aspartate receptor autoantibody levels in children with intractable and non-intractable epilepsy
Gunawan Prastiya Indra prastiya-i-g@fk.unair.ac.id
⁎
Widianti Nurani
Noviandi Riza
Samosir Sunny Mariana
Division of Neurology, Department of Child Health, Faculty of Medicine, Universitas Airlangga, Dr. Soetomo General Academic Hospital, Surabaya, Indonesia
⁎ Corresponding author. prastiya-i-g@fk.unair.ac.id
23 8 2024
15 9 2024
23 8 2024
10 17 e367347 5 2024
20 8 2024
21 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Objective

Intractable epilepsy in children is a prevalent neurological disorder that can pose serious risks. The involvement of the autoimmune system is a significant factor in the pathogenesis of the disease. The N-methyl-D-aspartate-receptor (NMDAR) is a glutamate receptor and ion channel present in neurons and is associated with the mechanism of autoimmune etiology in epilepsy. This study aims to compare the levels of NMDAR auto antibodies in children with intractable and non-intractable epilepsy.

Methods

A prospective analytic study was conducted from June to September 2022. The study sample consisted of patients aged 1 month to 18 years diagnosed with epilepsy and receiving anti-seizure medication (ASM) therapy at Dr. Soetomo General Academic Hospital, Surabaya. The patients were divided into two groups, namely intractable epilepsy and non-intractable epilepsy. The NMDAR autoantibody levels were determined using enzyme-linked immunosorbent assay (ELISA). Statistical analysis employed the chi-squared and Wilcoxon–Mann–Whitney tests.

Results

Seventy-five subjects were included in the study. Of these patients, 41.3 % with intractable epilepsy and 33.4 % with non-intractable epilepsy presented NMDAR auto antibodies. Analysis of the patient characteristics revealed a correlation between seizure frequency and NMDAR autoantibody positivity (P = 0.002) but not between the number of ASM and NMDAR autoantibody positivity (P > 0.05). The NMDAR autoantibody levels were not significantly different in children with intractable and non-intractable epilepsy (P = 0.157).

Conclusion

The NMDAR autoantibody levels were numerically higher in children with intractable epilepsy compared with children with non-intractable epilepsy.

Highlights

• The etiology of intractable epilepsy is diverse, one of them is considerable autoimmune process.

• NMDAR as an autoimmune marker is frequently identified in intractable epilepsy.

• In this study, NMDAR autoantibody is not correlated with intractable epilepsy in children.

Keywords

Autoimmune
Children
Disease
Intractable epilepsy
NMDAR
==== Body
pmc1 Introduction

Intractable epilepsy is characterized by the failure to administer two adequate therapies using anti-seizure medications (ASMs), either monotherapy or combination therapy, to achieve continued seizure freedom [1,2]. Based on scientific developments, intractable epilepsy is being increasingly associated with an autoimmune mechanism [3,4]. The evaluation of autoimmune-based neurological diseases includes diagnosis and therapy. In recent decades this field has developed rapidly, especially after the discovery of specific antibodies against neural and glial antigens. Molecular identification of these antigenic targets provides insight into the pathogenic mechanisms underlying various autoimmune-based neurological disorders, including epilepsy [[5], [6], [7]].

The recently recognised immune etiology concept applies to adults and children, as evidenced by autoimmune-related inflammation in the central nervous system [8,9]. Several auto antibodies have been detected in the serum of patients with epilepsy, including against the N-methyl-D-aspartate receptor (NMDAR) [10]. This receptor is associated with myriad neurological disorders, including epilepsy [11]. It is also believed to play a crucial role in synaptic plasticity control, as well as in mediating learning and memory functions [12].

The link between autoimmune processes and epilepsy has significant clinical implications, as immunotherapy administered as part of the treatment could lead to a clinical improvement in patients [6]. Autoimmune epilepsy often does not respond to conventional ASMs but does show a response to immunotherapy, underscoring its importance [13]. Early identification and detection of autoimmune epilepsy are critical because early intervention yields better outcomes [[14], [15], [16], [17]]. Therefore, this research aims to assess the levels of NMDAR auto antibodies in children with intractable epilepsy compared with those with non-intractable epilepsy.

2 Materials and methods

2.1 Design

This prospective analytic study assessed the levels of NMDAR auto antibodies in children diagnosed with intractable and non-intractable epilepsy. The study was conducted between June and September 2022 at the Pediatric Neurology Outpatient Installation, Department of Pediatrics, Dr. Soetomo General Academic Hospital, Surabaya.

2.2 Patients

The study sample comprised patients with intractable and non-intractable epilepsy who met the following inclusion criteria: aged 1 month to 18 years, had been diagnosed with either intractable or non-intractable epilepsy, received ASMs, and their parents or guardians provided informed consent to participate in the study. The patients were excluded if there was history of presence of other autoimmune diseases (e.g., type 1 diabetes mellitus, Crohn's disease, or Hashimoto thyroiditis), chronic inflammatory disorders, renal and hepatic impairments, cardiovascular and pulmonary conditions, neoplastic diseases, and received high-dose steroid therapy.

2.3 Ethics

The Health Research Ethics Committee of Dr. Soetomo General Academic Hospital, Surabaya, approved all study protocols on June 9, 2022, under an approval letter number 0430/KEPK/VI/2022.

2.4 Measurement

NMDAR auto antibodies in serum were measured at the Stem Cell Laboratory of the Institute of Tropical Diseases, employing the Assay Genie Human anti-NMDAR Antibody enzyme-linked immunosorbent assay (ELISA) Kit. Serum isolated from this blood was submitted to ELISA. The kit is stored at 2-8 °C. The NMDAR autoantibody examination method uses quantitative examination. NMDAR autoantibodies are examined using a 3 ml venous blood sample, then a 1000f sample were centrifuged for 10 min, then the serum is examined. Serum can be stored at −20 °C until −80 °C. The available microtiter plates are precoated with antigen. The sample placed in the well will bind to the antigen precoating. Washing is carried out to remove unbound reagents. Serum bound to antigen precoated will conjugate with horseradish peroxidase (HRP). In this quantitative colorimetric immunoassay, a specific antibody analogue binds to the target antigen. The absorbance is measured at 450 nm. The sensitivity of the assay is < 0.938 ng/ml, with a reading range of 1.563–100 ng/ml.

2.5 Statistical analysis

Data analysis was conducted using IBM SPSS Vers 23 Statistics. The chi-square test and the Wilcoxon–Mann–Whitney test were employed to determine whether there were significant differences between the intractable and non-intractable epilepsy groups; P < 0.05 was considered to indicate a statistically significant difference.

3 Results

A total of 75 children who met the inclusion and exclusion criteria were included in the study through simple random sampling. Data collection involved anamnesis, physical examination, supporting examinations, completion of data collection sheets, and blood sampling. The participants were divided into two groups: 37 children with intractable epilepsy and 38 with non-intractable epilepsy.

Table 1 presents the patients characteristics in the two groups. There were more male than female subjects in the intractable epilepsy group, while there was an equal number of males and females in the non-intractable epilepsy group. Among the different age categories, the highest proportion of patients was observed in the 1–5 years age group. The onset of seizures predominantly occurred in children under 1 year old in the intractable epilepsy group, whereas in the non-intractable epilepsy group, the highest occurrence was in the 1–5 years age group. General tonic-clonic seizures were the most common seizure type in both groups. Approximately a quarter of the children in the intractable epilepsy group experienced frequent seizures, while the majority of children in the non-intractable epilepsy group had infrequent seizures. More than a quarter of the patients in the intractable epilepsy group were using two or more ASMs, whereas common patients in the non-intractable epilepsy group were using one ASM. The duration of ASM therapy in both groups mostly ranged from 13 months to 5 years.Table 1 Patient characteristics.

Table 1Characteristic	Intractable epilepsy (n = 37)	Non-intractable epilepsy (n = 38)	P	
Sex, n (%)	
 Male	22 (29.4)	19 (25.3)	0.555	
 Female	15 (20.0)	19 (25.3)		
Age, n (%)	
 <1 year old	1 (1.3)	1 (1.3)	0.832	
 1–5 years old	16 (21.4)	13 (17.4)		
 6–10 years old	12 (16.0)	13 (17.4)		
 11–15 years old	6 (8.0)	10 (13.3)		
 >15 years old	2 (2.6)	1 (1.3)		
Early onset of seizures, n (%)	
 <1 year old	20 (26.6)	13 (17.4)	0.247	
 1–5 years old	11 (14.7)	16 (21.4)		
 6–10 years old	4 (5.3)	8 (10.7)		
 11–15 years old	1 (1.3)	1 (1.3)		
 >15 years old	1 (1.3)	0 (0.0)		
Seizure type, n (%)	
 General tonic-clonic	25 (33.3)	21 (28.0)	0.604	
 Partial	10 (13.3)	14 (18.7)		
 Atonic	2 (2.6)	3 (4.1)		
Seizure frequency, n (%)	
 Often	25 (33.3)	11 (14.7)	0.004a	
 Seldom	12 (16)	27 (36)		
Number of antiepileptic drugs, n (%)	
 1	17 (22.7)	25 (33.3)	0.000a	
≥2	20 (26.7)	13 (17.3)		
Duration of antiepileptic drug therapy, n (%)	
 <6 months	4 (5.3)	8 (10.7)	0.091	
 6–12 months	6 (8.0)	5 (6.6)		
 13 months–5 years	18 (24.0)	23 (30.7)		
 >5 years	9 (12.0)	2 (2.7)		
a Statistically significant based on the chi-square test.

Based on the chi-Square test, there was no significant difference in NMDAR autoantibody levels between children with intractable epilepsy and those with non-intractable epilepsy (χ2 = 0.127, P = 0.127) (Table 2). Similarly, the Wilcoxon Mann–Whitney test revealed no significant difference in NMDAR autoantibody levels between the two groups (W = 0.157, P = 0.1575) (Table 3).Table 2 Correlation between NMDAR autoantibody levels in children with intractable epilepsy and those with non-intractable epilepsy.

Table 2		Intractable epilepsy, n (%)	Non-intractable epilepsy, n (%)	P	
NMDAR autoantibody	Positive	31 (41.3)	25 (33.4)	0.127	
Negative	6 (8.0)	13 (17.3)	

Table 3 Comparison of NMDAR autoantibody levels in children with intractable epilepsy and those with non-intractable epilepsy.

Table 3NMDAR autoantibody (ng/ml)	Intractable epilepsy	Non-intractable epilepsy	P	
Range	0.58–43.72	0.37–65.08		
Median	6.49	4.53	0.157	

The chi-square (continuity correction) test indicated a significant correlation between seizure frequency and NMDAR autoantibody positivity in children with intractable and non-intractable epilepsy (χ2 = 0.002, P < 0.05) (Table 4). The strength of the correlation based on the contingency coefficient yielded a P-value of 0.360, indicating a moderately strong association between seizure frequency and NMDAR autoantibody positivity. On the other hand, there was not a significant correlation between the number of ASMs and NMDAR autoantibody positivity in children with intractable and non-intractable epilepsy (Fig. 1).Table 4 Correlation between seizure frequency and NMDAR autoantibody positivity in children with intractable epilepsy and those with non-intractable epilepsy.

Table 4		NMDAR autoantibody	P	C	
Positive, n (%)	Negative, n (%)	
Seizure Frequency	Often (>5 seizures in 6 months)	25 (69.4)	11 (30.6)	0.002	0.360	
Seldom (<5 seizures in 6 months)	12 (30.8)	27 (69.2)	

Fig. 1 Correlation between the number of administered ASMs and NMDAR autoantibody positivity in children with intractable epilepsy and those with non-intractable epilepsy.

Fig. 1

4 Discussion

Intractable epilepsy in children represent one of the most challenging aspects of pediatric neurology. This form of epilepsy is challenging to manage because it remains resistant to multiple ASMs. Males were known to have a higher risk of having epilepsy [18]. In this study, there were more males than females in the intractable epilepsy group and an equal number in the non-intractable epilepsy group. The incidence of intractable epilepsy in this center was 54 % [18]. In the present study, most of the patients with intractable epilepsy were in the 1–5 years age group (21.4 %), while there was an equal number of patients with non-intractable epilepsy in the 1–5 and 6–10 years age groups (17.4 %).

The majority of the children included in this study had intractable epilepsy, with the most common seizure types being general tonic-clonic seizures (33.3 %), followed by partial seizures (13.3 %) and atonic seizures (2.6 %). Andrianti [19] reported that among 103 children with epilepsy, the most common seizure type was general (55.3 %), followed by partial (37.9 %) and other types (6.8 %). Several studies have also shown that general seizures are the most common type, with Suwarba [20] reporting a prevalence of 80.4 % and Tjandrajani reporting a prevalence of 78.6 % for generalised seizures [21]. Otherwise Bintoro [22] found less than 8 % patients were generalised seizures.

In the present study there was a significant correlation between the number of ASMs and intractable epilepsy/non-intractable epilepsy (P < 0.05). The primary objective of treating patients with epilepsy with ASMs is to prevent seizures, to reduce their frequency, and to mitigate associated complications. The initial approach involves the administration of ASM monotherapy. In the present study, the most frequent duration of ASM therapy was 13 months to 5 years in the intractable (24 %) and non-intractable (30.7 %) epilepsy groups, with no significant difference between the groups. Choi et al. [23] reported that the use of ASMs for more than 12 months in patients with intractable epilepsy is due to the lack of clinical improvement in controlling seizures.

One of the etiologies of epilepsy is due to autoimmune. In this study, there was no significant difference in the NMDAR autoantibody levels in children with intractable epilepsy and non-intractable epilepsy. This finding deviates from the hypothesis that there is an association between autoimmune mechanisms and the etiology of intractable epilepsy. The presence of specific neuronal auto antibodies in intractable epilepsy is considered to be indicative of autoimmune epilepsy [24]. This notion aligns with a study that reported the presence of specific auto antibodies, including glutamic acid decarboxylase (GAD) in the serum of children with epilepsy [10].

The range of serum NMDAR auto antibodies was 0.37–65.08 ng/dl. The occurrence of recurrent seizures associated with elevated NMDAR autoantibody levels serves as an indicator of immune-mediated processes rather than pathogenic antibodies [25]. Antineuronal antibody production can be triggered by prolonged seizures, wherein inflammation-induced blood-brain barrier leakage leads to the presence of antineuronal antibodies in the blood. Studies have also investigated elevated antineuronal antibody levels in individuals with long-term epilepsy, suggesting its potential role in the pathogenesis of epilepsy, characterised by increased seizure severity and frequency [26]. Notably, patients with intractable epilepsy exhibited high levels of NMDAR auto antibodies, which provide evidence of an ongoing active immunological process and autoimmune etiology within the brain [27]. Furthermore, NMDAR auto antibodies have been detected in diseases suspected to be triggered by autoimmune mechanisms, such as diabetes mellitus, acute encephalitis, and glucose tolerance failure. Thus, in this study, patient with such comorbidity were excluded. Given their presence in these cases, it is plausible to observe elevated NMDAR autoantibody levels in patients with more than non-intractable epilepsy [28].

There was a moderately strong, significant correlation between seizure frequency and NMDAR autoantibody positivity in children with intractable and non-intractable epilepsy. The etiology of epilepsy plays a crucial role in influencing seizure frequency and guiding the management of patients with epilepsy. Among the various neuronal auto antibodies associated with epilepsy, the presence of NMDAR auto antibodies has been specifically linked to intractable epilepsy. These auto antibodies have been implicated in GABAnergic mechanisms that promote neuronal excitation, leading to an increased susceptibility to seizures. However, it should be noted that the clinical manifestation of intractable epilepsy can vary among individuals due to the diverse defence mechanisms employed against seizures. Such variations in NMDAR autoantibody levels contribute to the heterogeneity observed in the clinical presentation of patients with intractable epilepsy [29]. Kwan [1] found no significant differences between NMDAR autoantibody levels and seizure frequency or seizure duration. The four patients with intractable epilepsy had high NMDAR autoantibody levels; however, there was no correlation between the autoantibody levels and the duration of intractable seizures. These findings are consistent with the results reported by Peltola [29] and Falip [30]: no significant associations between NMDAR autoantibody levels and seizure frequency or duration.

In the present study, there was no association between the number of ASMs and NMDAR autoantibody positivity in children with intractable and non-intractable epilepsy. In a previous study, researchers examined NMDAR autoantibody levels in six patients with intractable epilepsy who underwent a 1-year treatment regimen consisting of two ASMs. Notably, there were no discernible differences in NMDAR autoantibody levels in relation to the duration of ASM therapy across all patients [31]. Another study investigating the evaluation of NMDAR autoantibody levels in five patients with juvenile myoclonic epilepsy demonstrated elevated levels of NMDAR auto antibodies. Nevertheless, there was no significant correlation between NMDAR autoantibody levels and the number of ASMs used or the duration of ASMs therapy [32]. Similarly, another study comparing two groups of patients diagnosed with epilepsy at an early stage and submitted to ASM therapy; the authors reported no significant difference in NMDAR autoantibody levels between the two groups [25].

The limitation of this study is that the NMDAR autoantibody levels were measured in blood serum and not cerebrospinal fluid due to ethical barriers limiting lumbar punctures in patients with epilepsy. In addition, many patients refuse lumbar puncture due to its invasiveness. The strength of this study is that it is the first to analyse NMDAR autoantibody levels in patients with intractable and non-intractable epilepsy. Regarding the higher incidence of intractable epilepsy, there is a need for specific diagnostic screening that allows management other than multi-ASM therapy. Additional research with other autoantibody markers that may be responsible for the pathogenesis of intractable epilepsy is needed.

5 Conclusions

NMDAR auto antibodies levels were numerically but not significantly higher in children with intractable epilepsy compared with those with non-intractable epilepsy. Further research is needed to find out more detail the pathogenesis of intractable epilepsy.

Ethical approval statement

The Health Research Ethics Committee of Dr. Soetomo General Academic Hospital, Surabaya, approved all study protocols on June 9, 2022, under an approval letter number 0430/KEPK/VI/2022.

Human experiments

Informed consent was obtained from all parents or guardians of subjects participating in this study to obtain more objective results.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data included in article. This research data is available from the corresponding author for further information requests.

CRediT authorship contribution statement

Prastiya Indra Gunawan: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Nurani Widianti: Writing – review & editing, Writing – original draft, Visualization, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Riza Noviandi: Writing – review & editing, Writing – original draft, Visualization, Project administration, Methodology, Formal analysis, Conceptualization. Sunny Mariana Samosir: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The author would like to thanks to pediatric neurology residents for helping data collection.
==== Refs
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