
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

72683
10.1038/s41598-024-72683-7
Article
Genotype-driven therapeutics in DEE and metabolic epilepsy: navigating treatment efficacy and drug resistance
Nguyen Yen Thi My 15
Vu Bao-Quoc 23
Nguyen Duy-Khai 4
Quach Ngoc-Vinh 4
Bui Liem Thanh 2
Hong Jeonghan 67
Bui Chi-Bao bcbao@medvnu.edu.vn

89
1 https://ror.org/00waaqh38 grid.444808.4 0000 0001 2037 434X Department of Biotechnology, International University, Vietnam National University Ho Chi Minh City, Ho Chi Minh City, Vietnam
2 https://ror.org/0071qz696 grid.25488.33 0000 0004 0643 0300 Institute of Food and Biotechnology, Can Tho University, Can Tho City, Vietnam
3 grid.444808.4 0000 0001 2037 434X Faculty of Computer Science, University of Information Technology, Vietnam National University Ho Chi Minh City, Ho Chi Minh City, Vietnam
4 Department of Neurology, City Children’s Hospital, Ho Chi Minh City, Vietnam
5 Unit of AI Genomics, DNA Medical Technology, Ho Chi Minh City, Vietnam
6 grid.264381.a 0000 0001 2181 989X Department of Medical Device Management and Research, Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University, Samsung Medical Center, Seoul, South Korea
7 HnB Genomics, Ulsan, South Korea
8 https://ror.org/00waaqh38 grid.444808.4 0000 0001 2037 434X University of Health Sciences, Vietnam National University Ho Chi Minh City, Ho Chi Minh, Vietnam
9 Unit of Molecular Biology, City Children’s Hospital, Ho Chi Minh City, Vietnam
16 9 2024
16 9 2024
2024
14 2160610 2 2024
10 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Neonatal intensive care unit (NICU), particularly in treating developmental and epileptic encephalopathy (DEE) and metabolic epilepsy (ME), requires a deep understanding of their complex etiologies and treatment responses. After excluding treatable cases such as infectious or autoimmune encephalitis, our focus shifted to a more challenging subgroup of 59 patients for in-depth genetic analysis using exome sequencing (ES). The ES analysis identified 40 genetic abnormalities, significantly including de novo variants. Notably, we found structural variation as duplications in regions 2q24.3, including SCN1A and SCN2A were observed in 7 cases. These genetic variants, impacting ion channels, glucose transport, transcription regulation, and kinases, play a crucial role in determining medication efficacy. More than one-third (34.2%) of patients with DEE had an unfavorable response to anti-seizure medications (ASMs) in the chronic phase. However, since the ketogenic supplementary diet showed a positive effect, more than three-quarters (80%) of these drug-resistant patients improved during a 3-month follow-up. In contrast, the ME had a lower adverse reaction rate of 9.1% (2/22) to specialized medications, yet there were 5 fatalities and 10 cases with unidentified genetic etiologies. This study suggests the potential of categorizing drug-resistant variants and that a ketogenic diet could be beneficial in managing DEE and ME. It also opens new perspectives on the mechanisms of the ketogenic diet on the discovered genetic variants.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72683-7.

Keywords

Clinical sequencing
DEE
Metabolic epilepsy
Drug response
Ketogenic diet
Subject terms

Genomics
Medical genetics
the Vietnam National Foundation for Science and Technology Development (NAFOSTED)IZVSZ3.203431 issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Neonatal intensive care unit (NICU) may experience seizures due to a wide range of etiologies, including acute brain injury1, stroke2 and encephalitis3,4, metabolic disturbances5 and rare developmental and epileptic encephalopathy (DEE)6. Accurate identification and targeted management of the underlying causes of these seizures are paramount in conjuntion with seizure control itself7. The selection of appropriate anti-seizure medications (ASMs) presents a complex challenge, particularly in cases of DEE, where seizures frequency exhibit drug resistance8. This resistance necessitates compels the adoption of more innovative therapeutic approaches, such as the ketogenic diet (KD)9, cannabidiol10, and cranial epilepsy neurosurgery11,12. Effective management of these complex cases often demands a collaborative, multidisciplinary approach13.

The integration of exome sequencing (ES) in the NICU practices marks a transformative shift in diagnostic methods, enabling a more profound understanding of the genetic factors driving these diseases5. Reflecting on our prior research on a DEE cohort from Vietnam, distinct genotype‒phenotype correlations emerged, offering insights into potential therapeutic avenues14. Nevertheless, gaps persist in our comprehension of variant-specific therapeutic responses and the influence of metabolic anomalies on clinical presentations within DEE cohorts. Notably, as highlighted by Sadleir, even two variants within the same gene can lead to varied clinical manifestations15, emphasizing the intricate interplay of functional dynamics in disease presentations and therapeutic complexities16,17. This challenge is further compounded by the fact that approximately one-third of patients do not respond optimally to ASMs, underscoring the urgency of refined diagnostic precision. In this evolving landscape, recent findings spotlighting genes such as CDKL5, KCNT1, PCDH19, SCN1A, SCN2A, SCN8A, SLC2A1, and STXBP1 hold promise, suggesting that tailored therapeutic interventions based on specific genetic variants are useful16,18. Nevertheless, the sporadic nature of genotype-phenotype correlations complicates the ability to predict therapeutic outcomes solely on genetic data. Expanding our understanding of ion channels, kinases, and transcription factors is crucial to unraveling the molecular complexities inherent in these neurological conditions. Despite the potential of ES in the neurological NICU, its full implications remain underexplored, necessitating greater collaboration between clinicians and geneticists to fully harness its benefits.

This study aims to investigate the role of ES in the neonatal neurological NICU, with a particular focus on its impact on the diagnosis and management of DEE and metabolic epilepsy (ME). Furthermore, it seeks to address the challenges associated with ES, from the interpretation of results to the management of incidental findings, with the overarching goal of optimizing patient outcomes.

Results

Patient distribution and treatment response in the NICU

A comprehensive review was conducted on 172 NICU patients, as outlined in the flow chart presented in Fig. 1, focusing on risk classification. In this group, most patients (113 in total) showed improvement and successfully controlled seizures caused by infectious or autoimmune encephalitis, all within seven days of initiating first-line treatment. These treatable patients were subsequently excluded from the genetic study group. Additionally, a subset of 59 patients with an average seizure age of onset of 2 years and 6 months, accounting for 34.3% of the total patients, were subjected to ES analysis and received ongoing monitoring via pediatric neurology. In this subset, the preliminary MRI diagnosis revealed specific abnormalities, such as hypoplasia of the corpus callosum, diffuse cortical dysplasia, polymicrogyria, focal cortical dysplasia, hippocampal sclerosis, corpus callosum agenesis, mild cortical atrophy, cortical dysplasia, generalized brain atrophy, cortical malformation, periventricular leukomalacia, and cases categorized as uncertain. The phenotypic breakdowns included likely Lennox-Gastaut syndrome (MIM#301058, 7 patients), Dravet syndrome (MIM#607208, 8 patients), Early infantile developmental and epileptic encephalopathy (EIDEE) previously known as Ohtahara syndrome (MIM#308350, 8 patients), Infantile epileptic spasm syndrome (IESS) or previously known as West syndrome (MIM#308350, 9 patients), and undefined phenotypes (5 patients) (Table 1). Moreover, a significant segment of this group (n = 22) was identified with ME, characterized by abnormal blood and CSF profiles; elevated lactate, ammonia, or atypical amino acid levels; and MRI evidence of cerebral atrophy or white matter anomalies (Table 2). EEG findings, while indicative of the respective diagnostic categories, presented overlapping features across different patients (Tables 1 and 2).

Table 1 Clinical feature of DEE group.

ID	A/S	Genetic variant	Inheritance	Reported/
Novel	Phenoty-pe	Drug
Response	Treatment	MRI findings	EEG findings	
1	3/M	SCN1A(NM_001165963.4):c.4942 C > T

p.(Arg1648Cys)

	De novo	Reported51	DS	Suboptimal	VPA, CBD	Mild cortical atrophy	Increased spike-wave activity	
2	1/F	SCN1B(NM_001037.5):c.254_255delGCinsAA

p.(Arg85Gln)

SCN1B(NM_001037.5):c.457G > A

p.(Asp153Asn)

	Paternal

Maternal

	Novel

Reported52

	ND	Suboptimal	VPA, CBD	Generalized brain atrophy	Burst suppression pattern	
3	2/M	STXBP1(NM_003165.6):c.589G > C

p.(Asp197His)

	De novo	Novel	ND	Partial	VPA	Uncertain	Focal epileptiform discharges	
4	2/M	CDKL5(NM_001323289.2):c.2341_2342delAGinsTA

p.(Arg781Ter)

	De novo	Novel	EIDEE	Resistant	PHB, ACTH	Diffuse cortical dysplasia	Normal	
5	3/M	SCN1A(NM_001165963.4):c.3976G > T

p.(Ala1326Ser)

	De novo	Novel	DS	Partial	VPA, CBD	Normal	Focal epileptiform discharges	
6	3/F	KCNQ2(NM_172107.4):c.1259 C > T

p.(Pro420Leu)

	De novo	Novel	LGS	Suboptimal	VPA, LMG, TPM, CBD	Normal	Normal	
7	3/M	SCN2A(NM_001040143.2):c.2557 C > T

p.(Arg853Trp)

	De novo	Novel	IESS	Suboptimal	ACTH, VGB, TPM	Cortical malformation	Increased spike-wave activity	
8	3/M	ARX(NM_139058.3):c.1112G > A

p.(Arg371Gln)

	De novo	Reported53	ND	Resistant	VPA, CBD	Hypoplasia of corpus callosum	Burst suppression pattern	
9	4/M	SCN1A(NM_001165963.4):c.2191_2192delAGinsCA

p.(Arg731Gln)

	De novo	Novel	DS	Partial	VPA, CBD	Normal	Normal	
10	4/F	GABRB3(NM_000814.6):c.613 A > G

(p.Arg205Gly)

	De novo	Novel	LGS	Complete	VPA, LMG, TPM, CBD	Normal	Normal	
11	4/F	STXBP1(NM_003165.6):c.728T > G

p.(Val243Gly)

	De novo	Novel	IESS	Partial	VGB, TPM	Periventricular leukomalacia	Hypsarrhythmia pattern	
12	5/F	SCN8A(NM_014191.4):c.2803_2804delGGinsTC

p.(Gly935Ser)

	De novo	Novel	ND	Resistant	VPA, CBD	Normal	Generalized spike-wave activity	
13	3/M	SCN1A(NM_001165963.4):c.559 C > T

p.(Arg187Trp)

	De novo	Novel	DS	Suboptimal	VPA, CBD	Cortical dysplasia	Normal	
14	3/M	KCNQ2(NM_172107.4):c.749T > C

p.(Val250Ala)

	De novo	Novel	EIDEE	Resistant	VPA, LMG, TPM, CBD	Corpus callosum agenesis	Burst suppression pattern	
15	2/F	SCN2A(NM_001040143.2):c.400 C > T

p.(Leu134Phe)

	De novo	Novel	IESS	Complete	ACTH, VGB, TPM	Uncertain	Increased spike-wave activity	
16	5/F	CDKL5(NM_001323289.2):c.2257 C > T

(p.Gln753Ter)

	De novo	Novel	EIDEE	Resistant	PHB, ACTH	Polymicrogyria	Normal	
17	2/F	SCN1A(NM_001165963.4):c.4408_4409delGGinsTC

p.(Gly1470Ser)

	De novo	Novel	DS	Partial	VPA, CBD	Normal	Focal epileptiform discharges	
18	5/M	GABRG2(NM_198904.4):c.644_645delGTinsAG

p.(Arg215Gln)

	De novo	Novel	LGS	Resistant	VPA, LMG, TPM, CBD	Normal	Generalized slow waves	
19	1/M	STXBP1(NM_003165.6):c.1450G > C

p.(Asp484His)

	De novo	Novel	IESS	Partial	VGB, TPM	Cortical malformation	Hypsarrhythmia pattern	
20	2/M	ARX(NM_139058.3):c.736_737delGAinsTG

p.(Asp246Cys)

	De novo	Novel	EIDEE	Resistant	PHB, ACTH	Normal	Burst suppression pattern	
21	5/F	SCN1A(NM_001165963.4):c.3905 A > G

p.(Asn1302Ser)

	De novo	Novel	DS	Complete	STP, VPA, CBD	Normal	Normal	
22	1/M	KCNQ2(NM_172107.4):c.637 C > G

p.(Arg213Gly)

	De novo	Novel	LGS	Suboptimal	VPA, LMG, TPM, CBD	Hippocampal sclerosis	Normal	
23	2/M	SCN2A(NM_001040143.2):c.2567G > A

p.(Arg856Gln)

	De novo	Reported54	IESS	Partial	ACTH, VGB, TPM	Cortical malformation	Increased spike-wave activity	
24	3/M	ARX(NM_139058.3):c.1096_1098delGACinsTAA

p.(Asp366Ter)

	De novo	Novel	EIDEE	Resistant	PHB, ACTH	Normal	Burst suppression pattern	
25	5/M	SCN1A(NM_001165963.4):c.2135G > A p.(Arg712Gln)	De novo	Novel	DS	Suboptimal	VPA, CBD	Normal	Normal	
26	4/F	GABRB3(NM_000814.6):c.580 C > G

p.(Arg194Gly)

	De novo	Novel	LGS	Partial	VPA, LMG, TPM, CBD	Focal cortical dysplasia	Generalized slow waves	
27	5/M	STXBP1(NM_003165.6):c.703 C > T

p.(Arg235Ter)

	De novo	Reported55	IESS	Partial	VGB, TPM	Periventricular leukomalacia	Hypsarrhythmia pattern	
28	5/M	SCN8A(NM_014191.4):c.2746 C > T

p.(Arg916Cys)

	De novo	Novel	ND	Suboptimal	VPA, CBD	Normal	Normal	
29	4/F	SCN1A(NM_001165963.4):c.574T > A

p.(Trp192Arg)

	De novo	Novel	DS	Complete	VPA, CBD	Cortical dysplasia	Focal epileptiform discharges	
30	4/M	KCNQ2(NM_172107.4):c.637 C > T

p.(Arg213Trp)

	De novo	Reported56	LGS	Suboptimal	VPA, LMG, TPM, CBD	Corpus callosum agenesis	Burst suppression pattern	
31	1/F	SCN1A: 2q24.3 (chr2:165985813–166128020) x4	De novo	Novel	EIDEE	Partial	PHB, ACTH	Generalized brain atrophy	Focal epileptiform discharges	
32	1/M	SCN1A: 2q24.3 (chr2:165985813–166128020) x4	De novo	Novel	IESS	Resistant	ACTH, VGB, TPM	Generalized brain atrophy	Focal epileptiform discharges	
33	1/F	SCN1A: 2q24.3 (chr2:165985813–166128020) x4	De novo	Novel	IESS	Resistant	ACTH, VGB, TPM	Generalized brain atrophy	Focal epileptiform discharges	
34	1/M	SCN2A: 2q24.3 (chr2:165294055–165392304) x3	De novo	Novel	IESS	Resistant	ACTH, VGB, TPM	Cortical dysplasia	Focal epileptiform discharges	
35	1/F	SCN2A: 2q24.3 (chr2:165294055–165392304) x3	De novo	Novel	EIDEE	Partial	PHB, ACTH	Cortical dysplasia	Focal epileptiform discharges	
36	1/F	SCN1A: 2q24.3 (chr2:165985813–166128020) x4	De novo	Novel	EIDEE	Partial	PHB, ACTH	Generalized brain atrophy	Focal epileptiform discharges	
37	1/M	SCN1A: 2q24.3 (chr2:165985813–166128020) x4	De novo	Novel	EIDEE	Resistant	PHB, ACTH	Generalized brain atrophy	Focal epileptiform discharges	
A/S: Age/Sex; F: Female, M: Male; CNV: Copy number variation; DS: Dravet syndrome, LGS: Lennox-Gastaut syndrome, EIDEE: Early infantile developmental and epileptic encephalopathy, IESS: Infantile epileptic spasms syndrome; STP: Stiripentol, VPA: Valproate, CBD: Cannabidiol, LMG: Lamotrigine, TPM: Topiramate, ACTH: Adrenocorticotropic hormone, VGB: Vigabatrin, PHB: Phenobarbital, CLZ: Clonazepam.

Table 2 Clinical feature of ME group.

ID	A/S	Genetic variant	Inheritance	Reported/
Novel	Phenotype	Drug
response	MRI Findings	EEG findings	
38	3y/M	SLC2A1:c.679 + 5G > T	De novo	Novel	GLUT1-DS, Elevated lactate in blood and CSF	Complete	Generalized cerebral atrophy	Interictal diffuse slow background activity, intermittent focal or generalized slowing	
39	3y/M	SLC2A1(NM_006516.4):c.399 C > A

p.(Cys133Ter)

	Maternal	Novel	GLUT1-DS, Elevated lactate in blood and CSF	Complete	Normal or non-specific abnormalities	Interictal diffuse slow background activity, intermittent focal or generalized slowing	
40	3y/M	ALDH7A1(NM_001182.5):c.1375 A > T p.(Ile459Phe)

ALDH7A1(NM_001182.5):c.608G > A

p.(Trp203Ter)

	Paternal

Paternal

	Novel

Novel

	PDE	Complete	Variable findings (cortical atrophy, atypical myelination, basal ganglia changes)	Burst-suppression pattern, multifocal epileptiform discharges	
41	4y/F	ALDH7A1(NM_001182.5):c.1003 C > T (p.Arg335Ter)

ALDH7A1(NM_001182.5):c.538dup (p.Glu180fsGlyfsTer48)

	Paternal

Paternal

	Reported57

Novel

	PDE	Complete	Variable findings (cortical atrophy, atypical myelination, basal ganglia changes)	Burst-suppression pattern, multifocal epileptiform discharges	
42	2y/F	POLG(NM_002693.3):c.1849_1851delCGTinsTGA

p.(Arg617Ter)

POLG(NM_002693.3):c.2740 A > C

p.(Thr914Pro)

	Paternal

Paternal

	Novel

Reported58

	AHS	Resistant	Progressive cerebral atrophy, basal ganglia changes	Slow background activity, multifocal or generalized epileptiform discharges	
43	2y/F	POLG(NM_002693.3):c.3104dup p.(Ser1036ValfsTer11)

POLG(NM_002693.3):c.424_425delCTinsGC

p.(Leu142Ala)

	Paternal

Paternal

	Novel

Novel

	AHS	Resistant	Progressive cerebral atrophy, basal ganglia changes	Slow background activity, multifocal or generalized epileptiform discharges	
44	1y/M	PDHA1(NM_000284.4):c.685_686delATinsTA

p.(Met229Ter)

	Maternal	Novel	PDCD, Elevated lactate in blood and CSF	Complete	Normal or non-specific abnormalities	Interictal slowing, multifocal or generalized epileptiform discharges	
45	2y/M	PDHA1(NM_000284.4):c.839T > G

p.(Ile280Ser)

	Maternal	Reported59	PDCD, Elevated lactate in blood and CSF	Complete	Normal or non-specific abnormalities	Interictal slowing, multifocal or generalized epileptiform discharges	
46	2y/F	Non-detected genetic etiology	-	-	Epilepsy with low glucose in blood, Elevated lactate in blood and CSF	Partial	Normal or non-specific abnormalities	Normal or non-specific abnormalities	
47	2y/F	Non-detected genetic etiology	-	-	Epilepsy with low glucose in blood, Elevated lactate in blood and CSF	Partial	Normal or non-specific abnormalities	Normal or non-specific abnormalities	
48	2y/F	Non-detected genetic etiology	-	-	Propionic acidemia: Elevated levels of propionic acid, 3-hydroxypropionic acid, methylcitric acid	Partial	Normal or non-specific abnormalities	Normal or non-specific abnormalities	
49	3y/F	Non-detected genetic etiology	-	-	Elevated levels of guanidinoacetate in blood or urine.	Complete	Variable findings (cortical atrophy, atypical myelination, basal ganglia changes)	Burst-suppression pattern, multifocal epileptiform discharges	
50	2y/M	Non-detected genetic etiology	-	-	Glycosylation Disorders	Complete	Generalized cerebral atrophy	Normal or non-specific abnormalities	
51	2y/M	Non-detected genetic etiology	-	-	Elevated ammonia in blood and/or urine, Elevated lactate in blood and CSF	Complete	White matter abnormalities	Normal or non-specific abnormalities	
52	2y/M	PCCB(NM_000532.5):c.1126 C > T

p.(Arg376Cys)

PCCB(NM_000532.5):c.1531dup p.(Thr511AsnfsTer8)

	Paternal

Paternal

	Reported60

Novel

	Propionic acidemia	Complete	Normal or non-specific abnormalities	Normal or non-specific abnormalities	
53	1 m /M	DNM1L(NM_012062.5):c.289 A > T

p.(Lys97Ter)

	De novo	Novel	Persistent lactic acidemia in blood and CSF	Deceased	ND	ND	
54	4y/F	SLC2A1(NM_006516.4):c.766 A > T

p.(Lys256Ter)

	De novo	Novel	Limb ataxia, GLUT1-DS	Complete	ND	Normal or non-specific abnormalities	
55	2y/F	Non-detected genetic etiology	-	-	Persistent lactic acidemia in blood and CSF	Deceased	ND	ND	
56	6 m/F	Non-detected genetic etiology	-	-	Episodically elevated lactate in blood and CSF	Deceased	ND	ND	
57	8 m/M	Non-detected genetic etiology	-	-	Persistent lactic acidemia in blood and CSF	Deceased	White matter abnormalities	ND	
58	1y/F	Non-detected genetic etiology	-	-	Episodically elevated lactate in blood and CSF	Deceased	White matter abnormalities	Normal or non-specific abnormalities	
59	2y6m/F	PGM1(NM_002633.3):c.43 C > T

p.(Gln15Ter)

PGM1(NM_002633.3):c.1324 A > G

p.(Met442Val)

	Paternal

Paternal

	Novel

Novel

	Congenital Disorder of Glycosylation, Type It	Complete	Normal or non-specific abnormalities	Normal or non-specific abnormalities	
A/S: Age/Sex, y: year, m: month; F: Female, M: Male; GLUT1-DS: Glucose transporter type 1 deficiency syndrome, PDE: Pyridoxine-dependent epilepsy, AHS: Alpers-Huttenlocher syndrome, PDCD: Pyruvate dehydrogenase complex deficiency; CSF: cerebrospinal fluid, ND: Non-determined.

Genetic variants in DEE

Three weeks after initiating exome sequencing, 17 genetic anomalies were identified, spanning the DEE spectrum. These variations predominantly included missense variants, with the rest being nonsense and frameshift variants, as illustrated in Fig. 2 and Table S1, and their protein domain and variation location are shown in Figure S1a–j. Notably, seven patients exhibited duplications at 2q24.3 regions, which are associated with the SCN1A and SCN2A regions, respectively (Figure S3a–b). In the DEE group, the variations spanned ten genes and fell into three functional categories: ion channels (75.7%), regulators (18.9%), and kinases (5.4%). A significant 93.5% of these variants were de novo variants, indicating sporadic occurrence. Of these variants, 87.1% were predicted to alter protein function, while the rest were protein truncating (12.9%). The channelopathies subgroup included 21 de novo variants across multiple genes, mostly missense variants (Tables S1 and Figure S2). Following the criteria established by the ACMG classification, we categorized these variants into distinct levels of pathogenicity, namely, pathogenic, likely pathogenic, variant of uncertain significance (VUS), and likely benign, as detailed in Table S1. The regulatory and kinase group included variants in the STXBP1, ARX, and CDKL5, featuring a mix of protein-truncating variants (PTVs) and protein-alternating variants (PAVs).

Genetic variants in the ME group

In the ME group, 45.5% of patients had no detectable genetic etiology. However, exome sequencing uncovered seven implicated genes in 54.5% of patients, revealing 18 genetic variants (Table S1) along with their respective protein domain and variation locations, as illustrated in Figure S1k–p. These were classified as likely pathogenic, pathogenic, or VUS, with in silico scores and inheritance patterns (de novo, paternal, maternal) detailed in Tables S1 and Figure S2.

Associations between genotype and ASMs

In the DEE group, we identified 12 genes associated with variable clinical presentations. Four ion channel-related variants from GABRB3, SCN1A, and SCN2A showed a positive response to prolonged treatment, while the remaining eight variants did not respond. Conversely, ARX variants, particularly in three male patients exhibiting an EEG pattern of burst suppression, were linked to ongoing seizures despite treatment with valproic acid (VPA), cannabidiol (CBD), and adrenocorticotropic hormone (ACTH). CDKL5 was noted in two patients with a phenotypic diagnosis of EIDEE; these patients were both resistant to phenobarbital (PHB) and ACTH. Variant in KCNQ2 such as c.749T > C; (p.Val250Ala) was identified in patients with Early infantile developmental and epileptic encephalopathies (EIDEE) who did not respond to combined VPA, lamotrigine (LMG), topiramate (TPM), or CBD treatment. Patient #12, with the SCN8A: c.2803_2804delGGinsTC; (p.Gly935Ser) variant, showed no significant improvement in response to VPA or CBD. A GABRG2: c.644_645delGTinsAG; (p.Arg215Gln) was also linked to a lack of response to combined VPA, LMG, TPM, and CBD.

In the ME group, most patients experienced seizure reduction or complementation with ASMs combined with a ketogenic, low-protein diet or special supplements. Two female patients with 2-year-old onset of disease were detected with heterozygous altered variants in the POLG (one with c.1849_1851delCGTinsTGA; (p.Arg617Ter) and c.2740 A > C; (p.Thr914Pro); one with p.Lys1035SerfsTer59 and c.424_425delCTinsGC; (p.Leu142Ala)) (Fig. 3; Table 2). MRI revealed progressive cerebral atrophy and basal ganglia changes, and the patients developed drug resistance and Alpers – Huttenlocher syndrome. Despite a good initial response to ASM for seizure control, Patient #53 with nonsense variant of DNM1L: c.289 A > T; (p.Lys97Ter) exhibited persistent lactic acidemia in blood and cerebrospinal fluid (CSF), and nonspecific abnormalities in EEG and MRI ultimately indicated an intractable drug response, followed by death (Table 2).

Supplementation with a ketogenic diet

The three-month assessment of the KD included 36 patients from DEE and ME groups, encompassing 20 males and 16 females, who continued to experience seizures despite receiving multiple ASMs. Only 2 patients (patients #34 and #37) were deemed ineligible for KD supplementation due to nutritional inadequacies. The data revealed that, in the DEE group, 59.1% (13/22) of the epilepsy patients experienced a shift in their response patterns when the KD was combined with multiple ASMs. Specifically, the addition of the KD benefited 36.4% (8/22) of the individuals, transitioning them from a drug-resistant state to a partial response. In contrast, 22.7% (5/22) of the patients (two patients with missense variants and three patients with duplications in SCN1A and SCN2A) who had a partial response to ASMs became increasingly worse and more resistant to the KD supplement (Fig. 3; Table 1). Within the ME group, 40% (2/5) of patients with Alpers-Huttenlocher syndrome exhibited a change in response pattern, transitioning from resistance to multiple ASMs therapies to a partial response when a KD supplement was added to their treatment regimen (Fig. 3).

Discussion

These findings align with previous studies on genetic epilepsy, reinforcing the validity and prevalence of genetic markers across diverse populations19. This consistency provides a notable diagnostic yield, particularly in identifying novel disease variants, including those associated with actionable drug genes that are crucial for precision medicine. Notably, we found that approximately 43.75% of the candidate genes were associated with channelopathies—disorders caused by dysfunctional ion channels. SCN1A emerged as a recurrently implicated gene in DEEs across various cohorts, including ours, highlighting its critical role in both diagnosis and treatment20–22 (Figure S3). Our study also highlighted the genetic heterogeneity underlying DEEs such as SCN2A, SCN1B, SCN8A, GABRG2, GABRG3 and KCNQ2 (variants are displayed in Figure S1 a—j). In addition, the findings revealed ARX, CDKL5, STXBP1, ALDH7A1, POLG, PDHA1, PCCB, DNM1L, and PGM1 (variants are displayed in Figure S1 k—p), suggesting the ongoing evolution of genomic knowledge and its impact on improving diagnostic precision.

Neonatal seizures present unique challenges, including variable causes, symptom severity, and patient responses. The current literature indicates the need for standardized criteria for evaluating antiseizure efficacy in newborns, complicating the optimization of treatment and dosage8. Yozawitz’s study also mentioned that when conventional antiseizure therapy fails to be controlled, clinicians could consider that the patients had abnormal metabolic findings causing ME. This was also reflected in our genetic cohort, in which the ES revealed that 20.3% (12/59) of the genetic cohort carried a genetically metabolic etiology. Based on genetic etiology, we categorized individuals with ME for whom KD therapy has shown benefits. Our records indicate that most of our patients had a favorable response to the combined regimen of ASMs and additional interventions such as KD, D-galactose, specific dietary plans, or specialized supplements. Thus, genetic testing should be considered when a genetic cause is suspected, as it can enable precision therapy approaches. Some genes may be strongly associated with typical phenotypes, providing clues about the underlying cause and potential pathogenic variants.

We observed a patient with the substitution variant of SCN8A, c.2803_2804delGGinsTC; (p.Gly935Ser) in transmembrane segment 2 (Figure S1d) who was resistant to sodium channel blockers (SCBs). Although classified as a variant of uncertain significance, this variant had an intolerant impact (Figure S5a) on protein stability (-1.13 of DDG score). This was suspected to be the reason for resistance to VPA and CBD23. SCN8A variants have been linked to gain-of-function (GoF) and loss-of-function (LoF) epilepsies24. Determining the specific action of such variants through functional validation is crucial for precision medicine.

Within our cohort, pathogenic PAVs in KCNQ2 exhibited a range of phenotypes. Most patients responded to SCBs (such as VPA, LMG, TPM, or CBD). However, one patient with the missense variant, c.749T > C; (p.Val250Ala) in KCNQ2 was resistant, suggesting that the location and functional status of the variant significantly influenced the drug response (Figure S4b and Figure S5b). It is hypothesized that ASMs bind to the pore region of channels and that variants in this area could impair binding, necessitating functional analyses to inform treatment. Previous findings have shown that ASMs likely target the pore region of channels, suggesting that variants in this vicinity could interfere with ASM binding25. Interestingly, these findings include a patient with a missense variant at the same Val250 position but different in altering amino acid, c.748G > C; (p.Val250Leu) who showed a positive response to ASMs, such as VPA and levetiracetam (LEV), suggesting these drugs for individuals with KCNQ2 variants25. The variability in treatment outcomes may be attributed to the specific functional implications of the variants. Notably, our finding that Val250Ala leads to a considerable reduction in protein stability (a DDG score of -1.87) contradicts that of Val250Leu, which causes only a minor decrease (DDG score of -0.57). The resistance observed with Val250Ala in response to SCBs raises suspicions of a gain-of-function phenotype in KCNQ2. Consequently, functional assays are advocated for exploring the disease mechanisms of Kv7.2 channels in the context of drug interactions.

GABRG2 encodes for heterotetrametric GABAA receptors consisting of extracellular N-terminal and transmembrane domains also shows variability in drug response. The patient with substitution variant, c.644_645delGTinsAG; (p.Arg215Gln) in GABRG2 did not respond to standard medication, supporting the hypothesis that novel variants can disrupt protein structure, affecting receptors26. At amino acid 215 (Figure S1f), the positively charged arginine was altered to neutrally charged glutamine, resulting in an unstable protein (-1.11 for the DDG score) (Figure S4c). As a result, we hypothesize that novel variants can disrupt the structural domain, affect surface tracking, and cause concentration of the subunit in the endoplasmic reticulum26,27. In addition, several variants, namely, Ala118Cys, Arg177Gly, and Ile218Ser, which are also located in the extracellular N-terminal domain, respond well to ASM, whereas Pro83Ser, Ile107Ter, and Ala106Ter exhibit drug resistance and penetrance27. Variants in the extracellular N-terminal domain usually respond well to ASM, while variants leading to resistance highlight the complexity of predicting and selecting appropriate therapies.

Previous reports have indicated that CDKL5 variants have been commonly found in females with early-onset seizures and is involved in DEE and WS28. The X-linked inheritance pattern has led to fewer reported instances of male carriers of CDKL5 variants, with some cases hinting at early male mortality. Our study revealed two unrelated pediatric cases of CDKL5-related diseases in which the nonsense mutations, c.2341_2342delAGinsTA; (p.Arg781Ter) and c.2257 C > T; (p.Gln753Ter) changed the structure and subsequently affected protein function (Figure S1i and Figure S4d). These patients resisted PHB and ACTH therapy, as did patients with severe anomalies in EEG and neuroimaging findings. This finding supports the notion that CDKL5 is a significant genetic contributor to neonatal seizures and profound developmental challenges. Consistent with the literature, CDKL5 variants tend to result in severe clinical manifestations in both sexes. Nonetheless, the severity of symptoms can vary by sex, as observed in a family with fraternal twins in which the male was more severely affected than the female was29. Additional reports corroborate this sex disparity, describing a cohort of males with CDKL5 variants who experienced refractory epilepsy, significant developmental delays, and adverse neurocognitive outcomes30.

ARX, which is vital for neuroblast proliferation and GABAergic neuron migration31, is linked to various phenotypes and often confers resistance to ASMs. These include X-linked West syndrome, X-linked myoclonic epilepsy with developmental dysplasia, Partington syndrome, and X-linked lissencephaly with ambiguous genitalia (XLAG)32. Additionally, brain malformations such as lissencephaly, agenesis of the corpus callosum, and midbrain malformations are commonly observed in male ARX individuals32. Our study identified three distinct ARX variants in male patients across two domains: in the homeodomain, a missense variant c.1112G > A; (p.Arg371Gln) and a nonsense variant c.1096_1098delGACinsTAA; (p.Asp366Ter); and in the acidic domain, a missense variant c.736_737delGAinsTG; (p.Asp246Cys) (Figure S1h). These variants, located in highly conserved areas, have an intolerant impact and may lead to abnormal protein function affecting DNA binding (Figure S4e and Figure S5d). This finding suggested that male patients with missense or nonsense variants in the homeodomain could have severe brain malformations and cognitive impairments and may respond poorly to drug treatments.

Copy number variants are known as a significantly recognized factor in the genetics of epilepsy. Through multiple large – scale studies, genomic “hotpots” associated with high-risk epilepsy have been detecting, including 1q21.1, 2q13, 9q34.3, 10q26.3, 15q11.2, 15q11-q13, 19q13.333–35. The result of CGH array revealed 2q24.3 microduplication involving SCN1A and SCN2A from 7 patients (Figure S4). SCN- gene on 2q24.3 duplication has been reported in a few cases. Marini’s study obsessed 2 cases related only SCN1A duplication at exon 8–16 and 26 with Dravet syndrome36. Although a duplication at multiple genes SCN2A, SCN3A, and SCN1A was early identified in patients with early onset developmental and epileptic encephalopathy from previous studies37,38, duplication at 2q24.3 solely involve SCN2A remain less explored. The latest case report found that a girl child with DEE and intractable seizure. Consistently, this study showed two patients identified SCN2A duplication with EIDEE and IESS associated with resistant/less response with multiple ASM therapy. However, after introducing a ketogenic diet, the patient with ASM resistance slightly reduce seizure frequency.

A KD, characterized by a low carbohydrate content and high fat content, is a primary treatment option for epilepsy that is resistant to conventional ASMs. Numerous retrospective and prospective studies substantiate its effectiveness, revealing that approximately 30% of young patients achieve complete seizure-free survival. In comparison, approximately 60% of patients experienced significant improvement, marked by a reduced seizure frequency of more than 50%39. Nonetheless, the application of KDs is limited owing to challenges associated with their execution, adherence to strict dietary regimens, and occurrence of adverse side effects40. The exact mechanisms through which the KD protects against epilepsy are still not fully understood, and there is a lack of clarity regarding the genetic factors that influence a patient’s responsiveness to the diet. With the development of modern genetic sequencing, KD has been considered the gold standard and should be prescribed early in the course of epilepsy-controlled treatment for several specific conditions, such as glucose transporter protein 1 deficiency syndrome (Glut1DS), pyruvate dehydrogenase deficiency (PDHD), EIDEE, CDKL5 encephalopathy, Lennox-Gastaut syndrome, Dravet syndrome, and EIDEE40. This finding is consistent with our study, which revealed that most patients exhibited effective responses against seizures after receiving the combination of a KD and ASMs. However, two patients, #5 and #9, were classified as having Dravet syndrome and became worse and more resistant to KD supplements combined with ASMs. Here, we present three patients, #8, #20, and #24, who underwent ACTH therapy but continued convulsions, which is consistent with the findings of previous studies41. Later, seizures did not appear after the patients were treated with KD or multiple ASM therapies (as summarized in Fig. 3).

Clear clinical and laboratory distinctions between DEE and ME patients facilitate their diagnosis and management. While myoclonic seizures typically suggest metabolic disorders, DEEs exhibit a wider variety of seizure types and primarily affect the nervous system42,43. ME often involves multiple organ systems and is indicated by distinctive laboratory findings, such as elevated lactate or ammonia levels42. Despite the precision of genetic analysis in guiding management, this approach has limitations, as evidenced by inconclusive results in some cases within the ME group. Nevertheless, when genetic testing identifies causative genes, as observed in three autosomal recessive cases, it enables precise genetic counseling. In DEEs, focused pharmacological treatment based on genetic understanding is vital for alleviating chronic symptoms and improving quality of life44. This personalized medical strategy, informed by genetic diagnostics, is increasingly critical in optimizing therapeutic efficacy and enhancing patient outcomes.

Method

Sample selection and recruitment

In 2022, a cohort of 172 patients with critical seizures, abnormal EEG and MRI findings, were recruited from the pediatric NICU at the City Children’s Hospital for a prospective cohort study. These patients were initially subjected to standard first-line treatments aimed at seizure control. Clinical diagnosis was typically performed through electroencephalography (EEG) and magnetic resonance imaging (MRI). To determine the underlying etiology of seizures caused by encephalitis, polymerase chain reaction (PCR) and autoimmune antibody testing were used to assess infectious and autoimmune encephalitis, respectively. Those who expressed negative results with these tests underwent ES. The primary focus of our study was to conduct ES on patients identified within the DEE and metabolic categories. The inclusion criterion was patients who had negative results for infectious and autoimmune encephalitis and were included in the ES study. Additionally, patients must have experienced developmental setbacks or a slowdown in progress coinciding with the start of epilepsy. Individuals should also display signs that might point to a metabolic issue, including intermittent episodes of worsening health, unusually large organs, or a distinctive smell. Moreover, laboratory tests should yield unusual results, such as atypical levels of amino acids or organic acids, or unusual enzyme activity that suggests a metabolic disorder. Exclusion criteria: Patients who have epilepsy due to incidents such as a head injury, brain deterioration, infections, or immune system disorders were not included in the study.

Exome sequencing and genetic analysis

Genetic variants were discovered through the ES to examine the genetic content of genomic DNA. Peripheral blood was collected in EDTA tubes, and DNA was extracted using the QIAamp DNA Blood Kit (QIAGEN, Singapore). The genomic DNA extract was followed by DNA purification using PureLink™ Genomic DNA (Invitrogen™, USA). The DNA concentration, measured using DropSense96, ranged from 80 to 200 ng/µL, with OD values (260/280) between 1.8 and 2.0 and by the Qubit 2.0 Fluorometer. The DNA samples were then fragmented, hybridized, and captured using the Illumina Exome panel. The libraries were assessed for enrichment by qPCR, and the size distribution and concentration were determined using the Agilent 2100 Bioanalyzer. Libraries prepared for sequencing were run on a NovaSeq 6000 Sequencing System by Macrogen (Republic of Korea) using 2 × 100 bp paired-end sequencing. Quality control of the raw sequence data, including Phred-score, GC content, read length, and sequence duplication levels, was conducted using FASTQC. The Trimmomatic tool was used for trimming and quality filtering of low-quality bases and adaptor sequences. The read pairs were subsequently aligned to the human reference genome (GRCh37/hg19) using BWA-MEM, followed by additional processing with MarkDuplicates and base quality score recalibration. Variant calling of SNPs and indels in VCF files were performed using the GATK Haplotype and GATK Variant Filtration filtered the data. ANNOVAR facilitated the identification of variants utilizing databases such as dbSNP, the Genome Aggregation Database (gnomAD), UCSC RefSeq, 1000G, and ESP6500. For bioinformatics analysis and variant interpretation, we followed our previous pipeline45, updated the ACMG criteria46 and gene panel47 to screen the disease–causing gene, including gnomAD v4.1.0 allele frequency and counts, ClinVAR reports, and VarSome 12.1.0 version48. The 3D structure of the protein was predicted using SWISS-MODEL49, and then the mutation points were modified in The PyMOL Molecular Graphics System, Version 3.0, Schrödinger, LLC. The rarity and pathogenicity of a variant were inferred from its frequency in the population, with rarer variants considered more pathogenic. Protein damage was predicted using in silico scores from tools such as DDG, SIFT, REVEL, CADD, Polyphen2, and MetaDome50.

Array CGH analysis

We utilized array CGH with the SurePrint G3 Human CGH Microarray Kit in 1 × 244 K (AMADID Number: 014693) (Agilent Technologies, Santa Clara, CA, USA). The test samples were labeled with cyanine 3-deoxyuridine triphosphate (Cy3-dUTP) via the SureTag DNA Labeling Kit (Agilent Technologies), while sex-matched reference DNA samples were tagged with Cy5-dUTP. Post-labeling, the DNA was purified and then mixed with Cot-1 DNA, a 10× array CGH blocking agent, and 2× HI-RPM hybridization buffer (Agilent Technologies). This mixture was then dispensed onto a microarray slide. Hybridization was conducted in an Agilent hybridization chamber set at 67 °C and 20 rpm for 24 h, followed by stringent washing with Agilent’s wash buffer 1 and wash buffer 2. Finally, microarray slide images were captured using the Agilent SureScan Microarray Scanner G2505C, enabling precise analysis of genome defects.

CNV analysis was conducted using Agilent Cytogenomics v5.2.0.2 and the human genome build hg18. CNVs were categorized as either gains or losses if the region contained at least three consecutive probes with a mean log2 ratio of ± 0.25. CNVs smaller than 300 kb were excluded from further analysis according to the guidelines for detecting pathogenic variants. To decode and validate the clinical significance of the identified CNVs, we tapped into a rich repository of public databases, including UCSC (http://genome.ucsc.edu, accessed in November 2023), OMIM (http://www.omim.org/), DECIPHER (http://decipher.sanger.ac.uk/), and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/). Our quest was to pinpoint pathogenic CNVs, both common and rare, syndromic and non-syndromic, by cross-referencing with previously documented pathogenic CNVs pertinent to DD/CM. Novel variants of uncertain significance (VUSs) were identified by exploring genomic alterations, including microdeletions and duplications. CNVs were classified as VUSs if the genes in the region had known functions but could not be directly associated with the disease under investigation.

Quantitative PCR

To validate the duplications of CNVs detected by array-CGH, we employed quantitative real-time PCR (qPCR). Primer sets were meticulously crafted for the selected genomic regions of target genes such as SCN3A, SCN2A, SCN1A, SCN9A, and the endogenous GAPDH gene as an internal control, utilizing Primer-3 Software (V.0.4.0). Each reaction was conducted in a 10 µL final volume, containing 5 µL of SYBR-Green qPCR master mix (DNA Medical Technology, HCMC, Vietnam), 10 pmol of each primer, and 20 ng of genomic DNA. The PCR reactions were performed in triplicate on a 96-well plate using the same SYBR-Green qPCR master mix. Data collection was executed with StepOne Plus™ Real-Time PCR Systems, and the raw data was processed using Data Assist software. Analysis of the qPCR results was carried out using the ∆∆CT method, with the final data visualization achieved through GraphPad PRISM software.

Assessment of treatment outcomes

The assessment of patient progress hinges on the frequency of seizures over three months. A ‘complete response’ was categorized as an absence of seizures or a minimal occurrence (≤ 2). A ‘partial response’ was noted when seizures significantly reduced, yet the count remained ≤ 10. A ‘suboptimal response’ was defined as a moderate reduction, with ≤ 20 seizure occurrences. In cases where seizure frequency remains unchanged, the condition is termed ‘resistant.’ Following the commencement of treatment, physicians meticulously document the patient’s progress and treatment efficacy through a standardized process, with a particular focus on the frequency of seizures. In cases where the primary treatment falls short of a complete response, the patient is subject to a comprehensive prediet evaluation by clinicians and nutritionists. This assessment encompasses a nutritional review, recording baseline weight and height, analysis of blood biochemical components such as serum lipids and albumin, and a urologic ultrasound to exclude any contraindications to initiating KD therapy. Based on these findings, we may advocate for incorporating a supplementary KD, in line with the established protocol9, which should be administered over an additional three-month period. During this extended phase, the medical team diligently observes the patient’s health, continually classifying the therapeutic outcomes using the predefined response categories.

Fig. 1 Flowchart outlining the study of pediatric NICU patients with seizures. A total of 172 patients underwent tests for autoimmune and infectious encephalitis. Positive patients were treated accordingly, while those testing negative were referred for genetic analysis via exome sequencing (ES), splitting them into ME and DEE groups. The results of ES are briefly represented as gene names with accompanying numbers in the following order: the first number indicates variants in drug-responsive cases, the second number specifies variants marked with an asterisk (*) for therapeutic resistance or ‘d’ for decreased response situations. Long-term outcomes, including seizure control, resistance, and mortality, were monitored. Some patients receive a KD as a supplement, especially those with a poor response to ASMs. Treatment success was assessed in relation to the patients’ genetic profiles.

Fig. 2 Variant types across genes for DEE and ME group with SCN1A shows the highest number of missense variants, while CDKL5 has a substantial number of both missense and nonsense variants. PGM1 showing mixed missense/nonsense and POLG having a notable frameshift presence.

Fig. 3 Overall treatment response by genetic variation. The Sankey diagram maps genetic variants to treatment responses, including ASMs and a KD. The left panel lists genes associated with seizure conditions. The middle panel shows ASM response categories to treatments: seizure-free, partial, resistant, suboptimal, and dead. The right side shows how these responses might change with the addition of a KD, with categories such as partial – KD and resistant – KD, suggesting partial or full resistance to treatment despite the diet. The width of the bands represents the number of patients exhibiting each treatment response per genetic variant.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

Supplementary Material 4

Supplementary Material 5

Supplementary Material 6

Supplementary Material 7

Supplementary Material 8

Acknowledgements

This research is funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number IZVSZ3.203431.

Author contributions

YTMN and B-QV drafted the article; acquired, analyzed and interpreted the data; DKN, N-VQ and N-TH contributed to the clinical and neurological ICU; acquired the data; JH and LTB critically revised the manuscript for important intellectual content and approved the submitted manuscript; C-BB conceived the project and design; acquired, analyzed and interpreted the data; and critically revised the manuscript for important intellectual content. All the authors approved the submitted manuscript.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

All methods involved in human research participants were performed in accordance with the guidelines andregulations of the Declaration of Helsinki. Informed consent was obtained from all subjects and their legalguardians. The study received Institutional Review Board (IRB) approval number #BVNDTP–2021-09-02 by City Children’s Hospital Ethics Committee. Researchers and healthcare professionals must take appropriatemeasures to protect the confidentiality of personal and medical information. The data should be anonymized ordeidentified, and no images can be obtained upon further request.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Yen Thi My Nguyen and Bao-Quoc Vu.
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References

1. Fordington S Manford M A Review of Seizures and Epilepsy Following Traumatic Brain Injury J. Neurol. 2020 267 3105 3111 10.1007/s00415-020-09926-w 32444981
Fordington, S. & Manford, M. A Review of Seizures and Epilepsy Following Traumatic Brain Injury. J. Neurol. 267, 3105–3111. 10.1007/s00415-020-09926-w (2020).32444981 10.1007/s00415-020-09926-w
2. Fox, C. K., Glass, H. C., Sidney, S., Lowenstein, D. H. & Fullerton, H. J. Acute seizures predict epilepsy after childhood stroke. 74, 249–256, doi: (2013). 10.1002/ana.23916
3. Kneen R Management of suspected viral encephalitis in children – association of british neurologists and british paediatric allergy, immunology and infection group national guidelines J. Infect. 2012 64 449 477 10.1016/j.jinf.2011.11.013 22120594
Kneen, R. et al. Management of suspected viral encephalitis in children – association of british neurologists and british paediatric allergy, immunology and infection group national guidelines. J. Infect. 64, 449–477. 10.1016/j.jinf.2011.11.013 (2012).22120594 10.1016/j.jinf.2011.11.013
4. Cellucci, T. et al. Clinical approach to the diagnosis of autoimmune encephalitis in the pediatric patient. 7, e663, doi:doi: (2020). 10.1212/NXI.0000000000000663
5. Perucca P Bahlo M Berkovic SF The genetics of epilepsy Annu. Rev. Genom. Hum. Genet. 2020 21 205 230 10.1146/annurev-genom-120219-074937
Perucca, P., Bahlo, M. & Berkovic, S. F. The genetics of epilepsy. Annu. Rev. Genom. Hum. Genet. 21, 205–230. 10.1146/annurev-genom-120219-074937 (2020).10.1146/annurev-genom-120219-074937
6. Allen AS De novo mutations in epileptic encephalopathies Nature 2013 501 217 221 10.1038/nature12439 23934111
Allen, A. S. et al. De novo mutations in epileptic encephalopathies. Nature 501, 217–221. 10.1038/nature12439 (2013).23934111 10.1038/nature12439
7. Costain G Cordeiro D Matviychuk D Mercimek-Andrews S Clinical application of targeted next-generation sequencing panels and whole exome sequencing in childhood epilepsy Neuroscience 2019 418 291 310 10.1016/j.neuroscience.2019.08.016 31487502
Costain, G., Cordeiro, D., Matviychuk, D. & Mercimek-Andrews, S. Clinical application of targeted next-generation sequencing panels and whole exome sequencing in childhood epilepsy. Neuroscience 418, 291–310. 10.1016/j.neuroscience.2019.08.016 (2019).31487502 10.1016/j.neuroscience.2019.08.016
8. Yozawitz, E., Neonatal & Seizures N. Engl. J. Med. 388, 1692–1700, doi:10.1056/NEJMra2300188 (2023).
9. Kossoff, E. H. et al. A Modified Atkins Diet is Effective for the Treatment of Intractable Pediatric Epilepsy. 47, 421–424, doi: (2006). 10.1111/j.1528-1167.2006.00438.x
10. Devinsky O Trial of cannabidiol for drug-resistant seizures in the dravet syndrome N. Engl. J. Med. 2017 376 2011 2020 10.1056/NEJMoa1611618 28538134
Devinsky, O. et al. Trial of cannabidiol for drug-resistant seizures in the dravet syndrome. N. Engl. J. Med. 376, 2011–2020. 10.1056/NEJMoa1611618 (2017).28538134 10.1056/NEJMoa1611618
11. Zhang L Hall M Lam SK Comparison of long-term survival with continued medical therapy, vagus nerve stimulation, and cranial epilepsy surgery in paediatric patients with drug-resistant epilepsy in the USA: An observational cohort study Lancet Child. Adolesc. Health 2023 7 455 462 10.1016/S2352-4642(23)00082-2 37276875
Zhang, L., Hall, M. & Lam, S. K. Comparison of long-term survival with continued medical therapy, vagus nerve stimulation, and cranial epilepsy surgery in paediatric patients with drug-resistant epilepsy in the USA: An observational cohort study. Lancet Child. Adolesc. Health 7, 455–462. 10.1016/S2352-4642(23)00082-2 (2023).37276875 10.1016/S2352-4642(23)00082-2
12. Dwivedi, R. et al. Surgery for drug-resistant epilepsy in children. 377, 1639–1647, doi: (2017). 10.1056/NEJMoa1615335
13. Li W The experience of the multidisciplinary team in epilepsy management from a resource-limited country Epilepsia open. 2019 4 85 91 10.1002/epi4.12290 30868118
Li, W. et al. The experience of the multidisciplinary team in epilepsy management from a resource-limited country. Epilepsia open. 4, 85–91. 10.1002/epi4.12290 (2019).30868118 10.1002/epi4.12290
14. Freed AS The impact of rapid exome sequencing on medical management of critically ill children J. Pediatr. 2020 226 202 212e201 10.1016/j.jpeds.2020.06.020 32553838
Freed, A. S. et al. The impact of rapid exome sequencing on medical management of critically ill children. J. Pediatr. 226, 202-212e201. 10.1016/j.jpeds.2020.06.020 (2020).32553838 10.1016/j.jpeds.2020.06.020
15. Sadleir LG Not all scn1a epileptic encephalopathies are dravet syndrome: early profound thr226met phenotype Neurology 2017 89 1035 1042 10.1212/wnl.0000000000004331 28794249
Sadleir, L. G. et al. Not all scn1a epileptic encephalopathies are dravet syndrome: early profound thr226met phenotype. Neurology 89, 1035–1042. 10.1212/wnl.0000000000004331 (2017).28794249 10.1212/wnl.0000000000004331
16. Bayat, A., Bayat, M., Rubboli, G. & Møller, R. S. Epilepsy syndromes in the first year of life and usefulness of genetic testing for precision therapy. 12, 1051 (2021).
17. Wolff, M. et al. Genetic and phenotypic heterogeneity suggest therapeutic implications in SCN2A-related disorders. J Brain 140, 1316–1336, (2017). 10.1093/brain/awx054.
18. Ohba, C. et al. Early Onset Epileptic Encephalopathy caused by de novo scn8a mutations. 55, 994–1000, doi: (2014). 10.1111/epi.12668
19. Hieu, N. L. T. et al. Genetic analysis using targeted exome sequencing of 53 vietnamese children with developmental and epileptic encephalopathies. 18810.1002/ajmg.a.62741 (2022).
20. Çapan Ö Yapıcı Y Özbil Z Çağlayan HS Exome data of developmental and epileptic encephalopathy patients reveals de novo and inherited pathologic variants in epilepsy-associated genes Seizure 2023 10.1016/j.seizure.2023.06.009 37977948
Çapan, Ö., Yapıcı, Y., Özbil, Z. & Çağlayan, H. S. Exome data of developmental and epileptic encephalopathy patients reveals de novo and inherited pathologic variants in epilepsy-associated genes. Seizure10.1016/j.seizure.2023.06.009 (2023).37977948 10.1016/j.seizure.2023.06.009
21. Do TTH SCN1A gene mutation and adaptive functioning in 18 Vietnamese children with Dravet syndrome J Clin Neurol 2017 13 62 70 10.3988/jcn.2017.13.1.62 28079314
Do, T. T. et al. SCN1A gene mutation and adaptive functioning in 18 Vietnamese children with Dravet syndrome. J. Clin. Neurol. 13, 62–70. 10.3988/jcn.2017.13.1.62 (2017).28079314 10.3988/jcn.2017.13.1.62
22. Symonds JD Incidence and phenotypes of childhood-onset genetic epilepsies: a prospective population-based national cohort Brain 2019 142 2303 2318 10.1093/brain/awz195 31302675
Symonds, J. D. et al. Incidence and phenotypes of childhood-onset genetic epilepsies: a prospective population-based national cohort. Brain 142, 2303–2318. 10.1093/brain/awz195 (2019).31302675 10.1093/brain/awz195
23. Brunklaus A Ellis R Reavey E Semsarian C Zuberi SM Genotype phenotype associations across the voltage-gated sodium channel family J. Med. Genet. 2014 51 650 658 10.1136/jmedgenet-2014-102608 25163687
Brunklaus, A., Ellis, R., Reavey, E., Semsarian, C. & Zuberi, S. M. Genotype phenotype associations across the voltage-gated sodium channel family. J. Med. Genet. 51, 650–658. 10.1136/jmedgenet-2014-102608 (2014).25163687 10.1136/jmedgenet-2014-102608
24. Fan HC Lee HF Chi CS SCN8A encephalopathy: case report and literature review Neurol. Int. 2021 13 143 150 10.3390/neurolint13020014 33915942
Fan, H. C., Lee, H. F. & Chi, C. S. SCN8A encephalopathy: case report and literature review. Neurol. Int. 13, 143–150. 10.3390/neurolint13020014 (2021).33915942 10.3390/neurolint13020014
25. Zhang Q Gene mutation analysis of 175 chinese patients with early-onset epileptic encephalopathy Clin. Genet. 2017 91 717 724 10.1111/cge.12901 27779742
Zhang, Q. et al. Gene mutation analysis of 175 chinese patients with early-onset epileptic encephalopathy. Clin. Genet. 91, 717–724. 10.1111/cge.12901 (2017).27779742 10.1111/cge.12901
26. Shen D De novo gabrg2 mutations associated with epileptic encephalopathies Brain 2017 140 49 67 10.1093/brain/aww272 27864268
Shen, D. et al. De novo gabrg2 mutations associated with epileptic encephalopathies. Brain 140, 49–67. 10.1093/brain/aww272 (2017).27864268 10.1093/brain/aww272
27. Komulainen-Ebrahim J Novel variants and phenotypes widen the phenotypic spectrum of gabrg2-related disorders Seizure 2019 69 99 104 10.1016/j.seizure.2019.03.010 31004928
Komulainen-Ebrahim, J. et al. Novel variants and phenotypes widen the phenotypic spectrum of gabrg2-related disorders. Seizure 69, 99–104. 10.1016/j.seizure.2019.03.010 (2019).31004928 10.1016/j.seizure.2019.03.010
28. Bodian, D. L., Schreiber, J. M., Vilboux, T., Khromykh, A. & Hauser, N. S. Mutation in an Alternative Transcript of CDKL5 in a boy with early-onset Seizures. Cold Spring Harbor Mol. case Stud. 410.1101/mcs.a002360 (2018).
29. Weaving LS Mutations of CDKL5 cause a severe neurodevelopmental disorder with infantile spasms and mental retardation Am. J. Hum. Genet. 2004 75 1079 1093 10.1086/426462 15492925
Weaving, L. S. et al. Mutations of CDKL5 cause a severe neurodevelopmental disorder with infantile spasms and mental retardation. Am. J. Hum. Genet. 75, 1079–1093. 10.1086/426462 (2004).15492925 10.1086/426462
30. Mirzaa GM CDKL5 and ARX mutations in males with early-onset epilepsy Pediatr. Neurol. 2013 48 367 377 10.1016/j.pediatrneurol.2012.12.030 23583054
Mirzaa, G. M. et al. CDKL5 and ARX mutations in males with early-onset epilepsy. Pediatr. Neurol. 48, 367–377. 10.1016/j.pediatrneurol.2012.12.030 (2013).23583054 10.1016/j.pediatrneurol.2012.12.030
31. Friocourt G Parnavelas JG Mutations in ARX result in several defects involving GABAergic neurons Front. Cell. Neurosci. 2010 10.3389/fncel.2010.00004 20300201
Friocourt, G. & Parnavelas, J. G. Mutations in ARX result in several defects involving GABAergic neurons. Front. Cell. Neurosci.10.3389/fncel.2010.00004 (2010).20300201 10.3389/fncel.2010.00004
32. Sherr EH The ARX story (epilepsy, mental retardation, autism, and cerebral malformations): one gene leads to many phenotypes Curr. Opin. Pediatr. 2003 15 567 571 10.1097/00008480-200312000-00004 14631200
Sherr, E. H. The ARX story (epilepsy, mental retardation, autism, and cerebral malformations): one gene leads to many phenotypes. Curr. Opin. Pediatr. 15, 567–571. 10.1097/00008480-200312000-00004 (2003).14631200 10.1097/00008480-200312000-00004
33. Montanucci L Genome-wide identification and phenotypic characterization of seizure-associated copy number variations in 741,075 individuals Nat. Commun. 2023 14 4392 10.1038/s41467-023-39539-6 37474567
Montanucci, L. et al. Genome-wide identification and phenotypic characterization of seizure-associated copy number variations in 741,075 individuals. Nat. Commun. 14, 4392. 10.1038/s41467-023-39539-6 (2023).37474567 10.1038/s41467-023-39539-6
34. Niestroj LM Epilepsy subtype-specific copy number burden observed in a genome-wide study of 17 458 subjects Brain 2020 143 2106 2118 10.1093/brain/awaa171 32568404
Niestroj, L. M. et al. Epilepsy subtype-specific copy number burden observed in a genome-wide study of 17 458 subjects. Brain 143, 2106–2118. 10.1093/brain/awaa171 (2020).32568404 10.1093/brain/awaa171
35. Olson H Copy number variation plays an important role in clinical epilepsy Ann. Neurol. 2014 75 943 958 10.1002/ana.24178 24811917
Olson, H. et al. Copy number variation plays an important role in clinical epilepsy. Ann. Neurol. 75, 943–958. 10.1002/ana.24178 (2014).24811917 10.1002/ana.24178
36. Marini C SCN1A duplications and deletions detected in dravet syndrome: implications for molecular diagnosis Epilepsia 2009 50 1670 1678 10.1111/j.1528-1167.2009.02013.x 19400878
Marini, C. et al. SCN1A duplications and deletions detected in dravet syndrome: implications for molecular diagnosis. Epilepsia 50, 1670–1678. 10.1111/j.1528-1167.2009.02013.x (2009).19400878 10.1111/j.1528-1167.2009.02013.x
37. Marilena V Infantile epilepsy associated with mosaic 2q24 duplication including scn2a and scn3a Seizure 2011 20 813 816 10.1016/j.seizure.2011.07.008 21893419
Marilena, V. et al. Infantile epilepsy associated with mosaic 2q24 duplication including scn2a and scn3a. Seizure 20, 813–816. 10.1016/j.seizure.2011.07.008 (2011).21893419 10.1016/j.seizure.2011.07.008
38. Takuya M Long-term course of early onset developmental and epileptic encephalopathy associated with 2q24.3 microduplication Epilepsy Behav. Rep. 2022 19 100547 10.1016/j.ebr.2022.100547 35733834
Takuya, M. et al. Long-term course of early onset developmental and epileptic encephalopathy associated with 2q24.3 microduplication. Epilepsy Behav. Rep. 19, 100547. 10.1016/j.ebr.2022.100547 (2022).35733834 10.1016/j.ebr.2022.100547
39. Neal EG The ketogenic diet for the treatment of childhood epilepsy: a randomised controlled trial Lancet Neurol. 2008 7 500 506 10.1016/s1474-4422(08)70092-9 18456557
Neal, E. G. et al. The ketogenic diet for the treatment of childhood epilepsy: a randomised controlled trial. Lancet Neurol. 7, 500–506. 10.1016/s1474-4422(08)70092-9 (2008).18456557 10.1016/s1474-4422(08)70092-9
40. Kossoff EH Optimal clinical management of children receiving dietary therapies for epilepsy: updated recommendations of the international ketogenic diet study group Epilepsia open. 2018 3 175 192 10.1002/epi4.12225 29881797
Kossoff, E. H. et al. Optimal clinical management of children receiving dietary therapies for epilepsy: updated recommendations of the international ketogenic diet study group. Epilepsia open. 3, 175–192. 10.1002/epi4.12225 (2018).29881797 10.1002/epi4.12225
41. Takeshita, Y. et al. Novel ARX Mutation Identified in Infantile Spasm Syndrome Patient. Hum. Genome Variation. 710.1038/s41439-020-0094-2 (2020).
42. Almannai M Al Mahmoud RA Mekki M El-Hattab AW Metabolic Seizures Front. Neurol. 2021 12 640371 10.3389/fneur.2021.640371 34295297
Almannai, M., Al Mahmoud, R. A., Mekki, M. & El-Hattab, A. W. Metabolic Seizures. Front. Neurol. 12, 640371. 10.3389/fneur.2021.640371 (2021).34295297 10.3389/fneur.2021.640371
43. Scheffer, I. E. et al. ILAE Classification of the epilepsies: position paper of the ILAE commission for classification and terminology. 58, 512–521, doi: (2017). 10.1111/epi.13709
44. Perucca E The pharmacological treatment of epilepsy: recent advances and future perspectives Acta Epileptologica 2021 3 22 10.1186/s42494-021-00055-z
Perucca, E. The pharmacological treatment of epilepsy: recent advances and future perspectives. Acta Epileptologica 3, 22. 10.1186/s42494-021-00055-z (2021).10.1186/s42494-021-00055-z
45. Phan ANL CD40LG mutations in vietnamese patients with x-linked hyper-igm syndrome; catastrophic anti-phospholipid syndrome as a new complication Mol. Genet. Genom. Med. 2021 9 e1732 10.1002/mgg3.1732
Phan, A. N. L. et al. CD40LG mutations in vietnamese patients with x-linked hyper-igm syndrome; catastrophic anti-phospholipid syndrome as a new complication. Mol. Genet. Genom. Med. 9, e1732. 10.1002/mgg3.1732 (2021).10.1002/mgg3.1732
46. Miller DT Recommendations for reporting of secondary findings in clinical exome and genome sequencing, 2021 update: a policy statement of the american college of medical genetics and genomics (acmg) Genet. Sci. 2021 23 1391 1398 10.1038/s41436-021-01171-4
Miller, D. T. et al. Recommendations for reporting of secondary findings in clinical exome and genome sequencing, 2021 update: a policy statement of the american college of medical genetics and genomics (acmg). Genet. Sci. 23, 1391–1398. 10.1038/s41436-021-01171-4 (2021).10.1038/s41436-021-01171-4
47. Zhang MW Epilepsy-associated genes: an update Seizure 2024 116 4 13 10.1016/j.seizure.2023.09.021 37777370
Zhang, M. W. et al. Epilepsy-associated genes: an update. Seizure 116, 4–13. 10.1016/j.seizure.2023.09.021 (2024).37777370 10.1016/j.seizure.2023.09.021
48. Kopanos C VarSome: the human genomic variant search engine Bioinformatics 2018 35 1978 1980 10.1093/bioinformatics/bty897
Kopanos, C. et al. VarSome: the human genomic variant search engine. Bioinformatics 35, 1978–1980. 10.1093/bioinformatics/bty897 (2018).10.1093/bioinformatics/bty897
49. Waterhouse A SWISS-MODEL: homology modelling of protein structures and complexes Nucleic Acids Res. 2018 46 W296 W303 10.1093/nar/gky427 29788355
Waterhouse, A. et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296–W303. 10.1093/nar/gky427 (2018).29788355 10.1093/nar/gky427
50. Wiel L MetaDome Pathogenicity analysis of genetic variants through aggregation of homologous human protein domains Hum. Mutat. 2019 40 1030 1038 10.1002/humu.23798 31116477
Wiel, L. et al. Pathogenicity analysis of genetic variants through aggregation of homologous human protein domains. Hum. Mutat. 40, 1030–1038. 10.1002/humu.23798 (2019).31116477 10.1002/humu.23798
51. Thompson CH Porter JC Kahlig KM Daniels MA George AL Jr. Nontruncating scn1a mutations associated with severe myoclonic epilepsy of infancy impair cell surface expression J. Biol. Chem. 2012 287 42001 42008 10.1074/jbc.M112.421883 23086956
Thompson, C. H., Porter, J. C., Kahlig, K. M., Daniels, M. A. & George, A. L. Jr. Nontruncating scn1a mutations associated with severe myoclonic epilepsy of infancy impair cell surface expression. J. Biol. Chem. 287, 42001–42008. 10.1074/jbc.M112.421883 (2012).23086956 10.1074/jbc.M112.421883
52. Watanabe H Mutations in sodium channel β1- and β2-subunits associated with atrial fibrillation Circ. Arrhythm. Electrophysiol. 2009 2 268 275 10.1161/circep.108.779181 19808477
Watanabe, H. et al. Mutations in sodium channel β1- and β2-subunits associated with atrial fibrillation. Circ. Arrhythm. Electrophysiol. 2, 268–275. 10.1161/circep.108.779181 (2009).19808477 10.1161/circep.108.779181
53. Thai, M. H. N. et al. Constraint and Conservation of paired-type Homeodomains Predicts the Clinical Outcome of Missense Variants of Uncertain Significance. 41, 1407–1424, doi: (2020). 10.1002/humu.24034
54. Møller RS Gene panel testing in epileptic encephalopathies and familial epilepsies Mol. Syndromol. 2016 7 210 219 10.1159/000448369 27781031
Møller, R. S. et al. Gene panel testing in epileptic encephalopathies and familial epilepsies. Mol. Syndromol. 7, 210–219. 10.1159/000448369 (2016).27781031 10.1159/000448369
55. Saitsu, H. et al. STXBP1 Mutations in Early Infantile Epileptic Encephalopathy with suppression-burst Pattern. 51, 2397–2405, doi: (2010). 10.1111/j.1528-1167.2010.02728.x
56. Sadewa, A. H. et al. Germ-line Mutation of KCNQ2, p.R213W, in a Japanese Family with Benign Familial Neonatal Convulsion. 50. 167-17110.1111/j.1442-200X.2008.02539.x (2008).
57. Jain-Ghai S Mishra N Hahn C Blaser S Mercimek-Mahmutoglu S Fetal onset ventriculomegaly and subependymal cysts in a pyridoxine dependent epilepsy patient Pediatrics 2014 133 e1092 1096 10.1542/peds.2013-1230 24664088
Jain-Ghai, S., Mishra, N., Hahn, C., Blaser, S. & Mercimek-Mahmutoglu, S. Fetal onset ventriculomegaly and subependymal cysts in a pyridoxine dependent epilepsy patient. Pediatrics 133, e1092-1096. 10.1542/peds.2013-1230 (2014).24664088 10.1542/peds.2013-1230
58. Rouzier C Quantitative multiplex pcr of short fluorescent fragments for the detection of large intragenic polg rearrangements in a large french cohort Eur. J. Hum. Genet. 2014 22 542 550 10.1038/ejhg.2013.171 23921535
Rouzier, C. et al. Quantitative multiplex pcr of short fluorescent fragments for the detection of large intragenic polg rearrangements in a large french cohort. Eur. J. Hum. Genet. 22, 542–550. 10.1038/ejhg.2013.171 (2014).23921535 10.1038/ejhg.2013.171
59. Egel RT Hoganson GE Katerji MA Borenstein MJ Zonisamide ameliorates symptoms of secondary paroxysmal dystonia Pediatr. Neurol. 2010 43 205 208 10.1016/j.pediatrneurol.2010.04.008 20691944
Egel, R. T., Hoganson, G. E., Katerji, M. A. & Borenstein, M. J. Zonisamide ameliorates symptoms of secondary paroxysmal dystonia. Pediatr. Neurol. 43, 205–208. 10.1016/j.pediatrneurol.2010.04.008 (2010).20691944 10.1016/j.pediatrneurol.2010.04.008
60. Nizon M Long-term neurological outcome of a cohort of 80 patients with classical organic acidurias Orphanet J. Rare Dis. 2013 8 148 10.1186/1750-1172-8-148 24059531
Nizon, M. et al. Long-term neurological outcome of a cohort of 80 patients with classical organic acidurias. Orphanet J. Rare Dis. 8, 148. 10.1186/1750-1172-8-148 (2013).24059531 10.1186/1750-1172-8-148
