Clinical characteristics and genetic analysis of patients with SCN1A gene pathological variant-related disorders: a single-center retrospective study

Abstract

Background

SCN1A Gene pathological variants can lead to a spectrum of epilepsy phenotypes ranging from mild genetic epilepsy with febrile seizures plus (GEFS+) to severe Dravet syndrome (DS). As most pathological variants are de novo variants, early prediction of phenotypes and prognosis is challenging, and the genotype-phenotype correlation remains unclear.

Method

We retrospectively analyzed the clinical and genetic data of 50 children with SCN1A variant-related epilepsy to explore the relationship between variant characteristics (location, in silico prediction scores), seizure characteristics, and phenotypes.

Results

This study included 50 children with SCN1A variant-related epilepsy, among whom 86.0% (43/50) had onset before the age of 1, 86.0% (43/50) exhibited fever sensitivity, and 68.0% (34/50) were accompanied by developmental delay; 36.0% (18/50) experienced status epilepticus(SE), 56.0% (28/50) had cluster seizures, and 48.0% (24/50) showed abnormal electroencephalogram(EEG) within one year of onset. Genetic analysis revealed that missense mutations accounted for 62.0% (31/50), with 78.0% (39/50) being de novo variants, and 24 previously unreported pathogenic variants were identified. The DS group (16 cases) had a significantly higher incidence of SE compared to the non-DS group (P = 0.041), while the non-DS group had a higher proportion of missense mutations (P = 0.014). Variant region analysis indicated that N-terminal variants were strongly associated with SE, and variants in the S2-S3/S3-S4 regions were prone to causing EEG abnormalities. In terms of treatment, the ketogenic diet was effective in 80.0% (4/5) of the children, while sodium channel blockers exacerbated seizures in 66.6% (4/6) of the children.

Conclusion

The genotype-phenotype correlation in SCN1A-related epilepsy is crucial for early diagnosis and management: missense mutations are more prevalent in non-DS phenotypes, while DS phenotypes carry a higher risk of SE. Previously unreported variant sites expand the genetic spectrum. Ketogenic diet (KD) may be an effective treatment option, and the use of Sodium channel blocker(SCB) requires caution.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13052-026-02269-8.

Introduction

The SCN1A gene encodes the Nav1.1 channel and is the most common pathogenic gene for epilepsy [1]. Its pathogenic variants are associated with various epilepsy syndromes, ranging from relatively mild phenotypes such as genetic epilepsy with febrile seizures plus (GEFS+) to severe Dravet syndrome (DS) [2, 3]. As a severe epilepsy syndrome, DS presents with diverse forms of epileptic seizures. Typically, children exhibit normal physical and psychomotor development at the onset of the first seizure, but gradually develop varying degrees of developmental delay within one year after the onset [4]. In terms of treatment, despite the use of multiple antiseizure medications (ASMs) and Ketogenic diet (KD), the prognosis for some children remains poor. Particularly, the use of Sodium channel blocker drugs(SCBs) may exacerbate epileptic seizures by blocking the function of sodium channels in inhibitory neurons, further unbalancing the excitation and inhibition within the brain [5]. To date, more than 2000 SCN1A Gene pathological variant sites have been identified, including missense mutations, frameshift mutations, and splice site mutations [6, 7]. Most disease-associated SCN1A variants are de novo, and research on their genotype-phenotype correlations is still insufficient. This study evaluated retrospective cohort data from 50 children with SCN1A-related epilepsy, investigating the relationships between seizure characteristics, variant types, locations, and computational scores with epilepsy phenotypes. We aim to further elucidate the associations among these features, which will assist clinicians in predicting the phenotypes and prognoses of patients with SCN1A-related epilepsy.

Materials and methods

Phenotypic and clinical data

This study is a retrospective analysis, collecting data from pediatric patients who were diagnosed with epilepsy caused by SCN1A Gene pathological variants through genetic testing at the Affiliated Children’s Hospital of Nanjing Medical University between September 2017 and September 2023. The research data encompass the genetic information of the pediatric patients, family history, characteristics of epilepsy onset (including gender, age at first onset, history of febrile seizures, triggers of onset, developmental delays, etc., with the last follow-up by the end of September 2023 ensuring a minimum of one year of follow-up for each patient), auxiliary examination results such as EEG and cranial magnetic resonance imaging (MRI), as well as the efficacy of ASM. All data were retrieved from the hospital’s Neusoft system and follow-up records.

The degree of developmental disability is assessed using the Gesell Developmental Schedules, and quantified by calculating the Developmental Quotient (DQ). Based on the DQ score, developmental disabilities are classified into three levels: mild (DQ 55–75), moderate (DQ 40–54), and severe (DQ < 40).This study diagnosed DS based on the criteria established by the International League Against Epilepsy (ILAE) in 2001, which specifically include: (1) a family history tendency of febrile seizures or epilepsy; (2) normal intellectual and motor development prior to onset; (3) onset within the first year of life, often triggered by fever; (4) initially normal EEG findings, followed by generalized, focal, or multifocal spike-and-wave or polyspike-and-wave discharges, with photosensitivity potentially appearing early; (5) normal mental and psychomotor development before the illness, but stagnation or regression in the second year, with possible neurological signs (such as ataxia, pyramidal signs); in addition, poor response to antiepileptic drug treatment.

Genetic testing

After obtaining informed consent from the guardians, 3 mL of peripheral venous blood was collected from the child and their parents. The IDT The xGen Exome Research Panel v2.0 whole exome capture chip was used for capture and sequencing to screen for pathological variant. The samples were analyzed and filtered through a precision diagnosis cloud platform system for genetic diseases, which integrates molecular biology annotation, biology, genetics, and clinical feature analysis. Combined with pathogenic variant databases, normal human genome databases, known clinical feature databases for 4,000 genetic diseases, and gene data analysis algorithms, hundreds of thousands of gene variants were classified. The variant classification was performed using a three-element classification system and the ACMG (American College of Medical Genetics and Genomics) gene variant classification system for gene database analysis. After Polymerase chain reaction (PCR) amplification of the target sequence, Sanger sequencing verification was performed using the ABI3730 sequencer, and the verification results were obtained through sequence analysis software. The pathological variant types were classified into missense mutations, nonsense mutations, frameshift mutations, non-frameshift mutations, and splice site mutations. De novo pathogenic variants were defined as those with negative parental gene testing.

Bioinformatics prediction and analysis

Use the following bioinformatics analysis websites to predict whether the missense mutation in the SCN1A gene of the affected child affects protein function:.

Mutation Taster: https://www.mutationtaster.org/;

Polymorphism Phenotyping v2 (PolyPhen_2): http://genetics.bwh.harvard.edu/pph2/;

Provean (https://www.jcvi.org/research/provean/);

FunNC (https://funnc.shinyapps.io/shinyappweb/) [8].

The aforementioned tools are merely one of the many tools used for predicting the functional impact of missense variants, and the results obtained have limitations.

Statistical processing

Statistical analysis was performed using SPSS 26.0 for data analysis. Categorical variables were analyzed using the chi-square test or Fisher’s exact test, while continuous variables were analyzed using the t-test or Mann-Whitney U test. A P-value of < 0.05 was considered statistically significant.

Ethical approval

All researchers signed informed consent forms after they or their guardians fully understood the content of this study. The retrospective review of the research data in this article was approved by the Ethics Review Committee of our hospital (Reference Number: 202401016-1).

Result

Queue characteristics

Among the 50 children with SCN1A pathological variants, males accounted for 56.0%, and females accounted for 44.0%. Among them, 43 cases (86.0%) developed the condition within the first year of life, with the earliest onset at 2 months and the latest at 3 years of age (Table 1). Developmental impairments of varying degrees were observed in 34 cases (68.0%) one year after onset. In 32 cases (64.0%), the first episode was accompanied by fever. Regarding triggers, 43 cases (86.0%) exhibited heat sensitivity, 4 cases (8.0%) had a history of episodes following vaccination, and a few triggers included photosensitivity and excitement. Analysis of seizure types revealed a diverse spectrum of epilepsy phenotypes within the cohort. GTCS were the most prevalent, observed in 24 patients (48.0%), followed by FOS in 15 patients (30.0%). Other seizure types included MS in 6 patients (12.0%), sGCS in 9 patients (18.0%), and aAS in 3 patients (6.0%). Many patients experienced multiple seizure types, which is a hallmark of DS.Among the 50 cases, 18 (36.0%) experienced status epilepticus(SE), and 28 (56.0%) had a history of cluster seizures. All children underwent EEG and MRI examinations. Only about half (24 cases) of the children showed abnormal EEG findings within the first year of onset, with background slowing being the most common, observed in 19 cases (Table 2). MRI results revealed no significant abnormalities in 30 cases, while the remaining 20 cases exhibited nonspecific changes, commonly including widened extracerebral spaces and fullness of the lateral ventricles.

Table 1.

Clinical characteristics of patients with SCN1A pathogenic variants

Patient	Sex	Fever of first seizure	Age of onset	heat sensitivity	Developmental delay	Status epilepticus	Seizure type	Treatment	Sensitive treatment	Family history	Inducement	Cluster seizures	MRI	EEG	Epilepsy first year EEG abnormalities
1	Boy	No	3 m	Yes	Moderate	No	sGCS	VPA, TPM, KD	KD	Yes	Bathing, fever	No	Normal	Spike-and-slow-wave / sharp-and-slow-wave	Yes
2	Boy	Yes	5 m	Yes	Severe	Yes	aAS	VPA, CZP, LEV, TPM, PER		No	Fever	No	Normal	Background slowing, sharp and slow waves/small sharp waves	No
3	Girl	No	6 m	Yes	Severe	Yes	GTCS、MS	LEV, VPA, TPM	VPA	No	Fever	Yes	Vascular abnormalities accompanied by peripheral demyelinating changes	Background slightly slow, sharp slow waves/fast waves	Yes
4	Girl	Yes	6 m	Yes	No	No	GTCS	LEV, VPA	VPA	Yes	Fever	Yes	Normal	Spike-and-slow-wave	No
5	Boy	No	6 m	Yes	Moderate	No	GTCS、FOS	VPA, TPM	TPM	No	Fever	Yes	Normal	Background slowing, sharp waves/sharp and slow waves	Yes
6	Girl	Yes	10 m	Yes	No	No	GTCS	VPA, TPM	TPM	No	Fever	No	Normal	Background slowing, slow waves/small spike-and-slow waves	No
7	Boy	No	15 m	No	Mild	No	GTCS	VPA	VPA	Yes	No	Yes	Normal	Normal	No
8	Girl	Yes	5 m	Yes	Mild	Yes	GTCS	VPA, TPM	TPM	No	Fever, vaccination.	No	Normal	Normal	No
9	Boy	No	6 m	No	No	Yes	GTCS、FOS	VPA, KD	KD	No	No	No	Normal	Small spike and slow wave	No
10	Boy	No	5y	No	No	No	GTCS	OXC	OXC	Yes	No	Yes	Normal	Spike slow wave/Sharp slow wave	Yes
11	Girl	Yes	11 m	Yes	Mild	No	GTCS	VPA	VPA	Yes	Bathing, fever	No	Normal	Small sharp slow wave	No
12	Girl	No	10 m	Yes	No	No	GTCS、FOS	LEV, VPA, TPM	VPA, TPM	No	Fever	Yes	Normal	Background slowing down	Yes
13	Girl	Yes	7 m	Yes	Severe	Yes	FOS	VPA, LEV, CLB		No	Bathing, fever	Yes	Normal	Spike and slow wave	Yes
14	Boy	Yes	4 m	Yes	Moderate	No	GTCS	VPA, TPM, CLB		No	Bathing, fever	Yes	Normal	Normal	No
15	Girl	No	3y	No	Mild	No	GTCS	VPA, CZP	VPA, CZP	Yes	No	No	Normal	Background slowdown, spike slow wave/multi-spike slow wave/slow wave	No
16	Boy	Yes	6 m	Yes	Moderate	No	GTCS	VPA, LEV, TPM	VPA, LEV	Yes	Bathing, fever	No	Normal	Background slowing down	No
17	Boy	Yes	10 m	Yes	Moderate	No	GTCS、FOS	VPA, LEV, TPM, KD		No	Fever	Yes	Normal	Slow wave/Spike slow wave	No
18	Boy	Yes	9 m	Yes	Mild	No	FOS	VPA, LEV, CZP, TPM	TPM	No	Fever	No	Normal	Background slows down, spike wave/spike slow wave	Yes
19	Girl	Yes	3 m	Yes	Moderate	No	GTCS、FOS	VPA, LEV, TPM, CLB	TPM, CLB	No	Fever, vaccination	Yes	Normal	Normal	No
20	Girl	Yes	< 1y	Yes	Mild	No	GTCS、FOS、aAS	VPA, LEV	VPA	Yes	Fever	No	Normal	Small spines and slow waves	No
21	Girl	No	7 m	Yes	No	Yes	sGCS	VPA, TPM, CZP, KD, CLB	CZP, KD	No	Fever	No	Normal	Spike slow/Sharp slow wave	Yes
22	Girl	No	8 m	Yes	Mild	No	sGCS	LEV	LEV	No	Fever	No	Normal	Normal	No
23	Boy	No	4 m	Yes	Mild	Yes	sGCS	VPA	VPA	Yes	Fever, bathing, excitement	Yes	Normal	Normal	Yes
24	Boy	No	11 m	Yes	Moderate	No	sGCS	OXC, LTG, VPA, LEV, TPM	VPA, LEV, TPM	No	Fever	Yes	Normal	Background slowing, spike-and-slow waves	No
25	Boy	Yes	6 m	Yes	No	Yes	FOS	VPA, LEV, KD	KD	No	Fever, bathing	Yes	Normal	Small spike wave	No
26	Boy	No	2 m	Yes	Moderate	No	GTCS、MS	VPA, TPM	TPM	No	Fever, bathing	No	Mild cerebral atrophy-like changes	Background slowing, slow waves/polyspike slow waves	No
27	Boy	No	3 m	Yes	Moderate	No	sGCS	OXC, VPA, TPM, CLB		No	Bathing, fever	Yes	Normal	Polyspike-and-slow-wave/Spike-wave	Yes
28	Girl	No	8 m	No	No	Yes	GTCS	VPA, TPM, LEV	VPA, TPM, LEV	No	No	No	Normal	Background slowdown	Yes
29	Girl	Yes	9 m	Yes	Mild	No	GTCS	LEV		No	Fever	Yes	Normal	Normal	No
30	Boy	Yes	9 m	Yes	No	Yes	MS	VPA	VPA	Yes	Fever, bathing	Yes	Normal	Small spike wave	No
31	Girl	Yes	14 m	No	Mild	No	sGCS	VPA	VPA	No	No	No	Corpus callosum dysplasia, bilateral periventricular heterotopia	Sharp wave/sharp and slow wave/spike wave/spike and slow wave	Yes
32	Boy	Yes	11 m	Yes	No	No	GTCS、aAS	VPA, LEV, TPM	TPM	Yes	Fever	Yes	Normal	Sharp wave/sharp and slow wave/spike wave/spike and slow wave	Yes
33	Boy	Yes	8 m	Yes	No	No	GTCS、FOS	LEV, VPA	VPA	Yes	Fever, bathing	Yes	Normal	Normal	No
34	Girl	Yes	5 m	Yes	Moderate	No	GTCS	VPA, TPM	VPA, TPM	No	Fever, bathing	Yes	Normal	Normal	No
35	Boy	Yes	4 m	Yes	Mild	No	MS	VPA, TPM		No	Fever, bathing, excitement	Yes	Normal	Spike-and-slow wave/slow wave	Yes
36	Girl	Yes	< 1y	Yes	NO	No	MS	VPA		No	Fever	No	Normal	Background activity slowing, spike-and-slow wave/polyspike-and-slow wave	Yes
37	Girl	Yes	22 m	Yes	No	No	AS			Yes	Fever	No	Normal	Normal	No
38	Boy	Yes	4 m	Yes	Severe	Yes		LEV, TPM, CLB		No	Fever	No	Normal	Normal	No
39	Boy	Yes	9 m	Yes	Severe	Yes	FOS	VPA, OXC, TPM		No	Fever	No	Normal	Slow wave / spike-and-slow wave	No
40	Boy	No	7 m	Yes	Mild	No	sGCS	VPA, LEV, TPM		No	Fever	Yes	Normal	Background slowing, sharp waves/sharp and slow waves	Yes
41	Boy	No	4 m	Yes	Moderate	Yes	GTCS	OXC, VPA, CLB		No	Fever	Yes	Normal	Background slowing, spikes/spike-and-slow waves	Yes
42	Girl	Yes	8 m	Yes	Severe	Yes	FOS	LEV, VPA, TPM, CLB	CLB	No	Fever	No	Normal	Background slowing, spike-and-slow wave/polyspike-and-slow wave/spike	Yes
43	Boy	Yes	8 m	Yes	Severe	Yes	GTCS	VPA, TPM		Yes	Fever	Yes	Pineal gland cystic changes, brain atrophy-like changes	Background slowing, spike-and-slow waves	Yes
44	Boy	Yes	14 m	Yes	No	No	GTCS	VPA	VPA	No	Fever	Yes	Normal	Sharp and slow waves / Multiple sharp waves / Sharp slow waves	Yes
45	Girl	Yes	6 m	Yes	No	Yes	AS	VPA, LEV	LEV	Yes	Fever	Yes	Normal	Background slightly slow, small sharp (slow) waves	No
46	Boy	Yes	7 m	Yes	Mild	Yes	FOS	VPA		No	Fever, vaccination	No	Normal	Background activity slows down	Yes
47	Girl	No	5 m	No	Severe	Yes	FOS	VPA, TPM, OXC, PER, LCM, LTG, CLB	TPM, PER, CLB	Yes	No	Yes	Normal	Irregular slow wave	Yes
48	Girl	Yes	8 m	Yes	Mild	No	sGCS	LEV, VPA	LEV, VPA	No	Fever, vaccination, bathing.	Yes	Normal	Normal	No
49	Boy	Yes	4 m	Yes	Mild	No	FOS	LEV, VPA	VPA	No	Fever	Yes	Normal	Irregular spikes/spike-and-slow waves	Yes
50	Boy	Yes	3y	Yes	No	No	MS	LEV	LEV	No	Fever	No	Normal	Sharp and slow waves / Asynchronous sharp waves	Yes

Note: m=month; y= year; sGCS, secondary generalized clonic seizure; aAS, atypical absence seizure; MS, myoclonic seizures; GTCS, generalized tonic-clonic seizure; FOS, focal seizure; VPA, valproate; LEV, levetiracetam; TPM, topiramate; PER, perampanet; OXC, oxcarbazepine; CZP, clonazepam; LCM, lacosamide; CLB, clobazam; LTG, lamotrigine; Cluster seizures: Two or more epileptic seizures within 24 h; Status epilepticus is defined as a single epileptic seizure lasting for ≥ 30 min, or frequent seizures with no recovery of consciousness during the interictal period, lasting for ≥ 30 min

Table 2.

Summary of clinical data of 50 patients

Demographics	Total cohort(n = 50)
Characteristic	n	(%)
Sex
Female	22	(44.0%)
Male	28	(56.0%)
Age at seizure onset
<1y	43	(86.0%)
≥ 1y	7	(14.0%)
Cluster seizures
Yes	28	(56.0%)
No	22	(44.0%)
Positive family history
Yes	16	(32.0%)
No	34	(68.0%)
Dravet syndrome
Yes	16	(32.0%)
No	34	(68.0%)
Fever of first seizure
Yes	32	(64.0%)
No	18	(36.0%)
Thermal sensitivity
Yes	43	(86.0%)
No	7	(14.0%)
Status epilepticus
Yes	18	(36.0%)
No	32	(64.0%)
Developmental delay
Yes	34	(68.0%)
No	16	(32.0%)
EEG background slows down
Yes	19	(38.0%)
No	31	(62.0%)
Epilepsy first year EEG
Abnormal	24	(48.0%)
Normal	26	(52.0%)
Ketogenic diet
Effective	4	(80.0%)
No effect	1	(20.0%)
ASMs
No drugs	1	(2.0%)
Single drug	12	(24.0%)
Two kinds	15	(30.0%)
≥ 3 kinds	22	(44.0%)
Therapeutic response
Seizure reduction ≥ 50%	38	(76.0%)
Seizure reduction<50%	12	(24.0%)

Pathogenic variant score

This study analyzed 50 pediatric epilepsy patients with SCN1A Gene pathological variants. The results showed that missense mutations were the most common, accounting for 31 cases (62.0%). Other mutation types included frameshift mutations, splice site mutations, nonsense mutations, insertion mutations, and deletion mutations(Table 3). Among these, 10 cases (20.0%) were inherited variants, while 39 cases (78.0%) were de novo variants. Due to the unavailability of the mother’s sample for one patient, the origin of the pathological variant site could not be determined. Evolutionary conservation analysis revealed that all missense mutation sites are highly conserved (Supplementary Fig. 1), suggesting their critical role in Nav1.1 channel function. We employed multiple software tools to predict the pathogenicity of 31 missense mutations: Mutation Taster predicted all 31 missense mutations as pathogenic; PolyPhen_2 predicted 25 cases (80.6%) as likely pathogenic, 4 cases (12.9%) as possibly pathogenic, and 2 cases (6.5%) as benign; Provean predicted 30 cases (96.7%) as pathogenic and 1 case (3.2%) as neutral.We performed functional classification of 31 missense variants using FunNC and compared the distribution of loss-of-function (LOF) or gain-of-function (GOF), and “no functional impact” categories between DS and non-DS individuals using Fisher’s exact test (Supplementary Table 1). Although the frequencies of LOF and GOF variants did not differ significantly between the two groups, we observed that all variants predicted to have “no functional impact” (4/4) occurred exclusively in the non-DS group, and this group difference was statistically significant (P = 0.046). These findings suggest a potentially structured distribution pattern of predicted functional consequences across clinical subtypes.However, given the limited number of variants within this classification, the results should be interpreted with caution.

Table 3.

Statistical Analysis of clinical and genetic characteristics of children with Dravet syndrome and those without Dravet syndrome

Case	Nucleotide variation	Amino acid changes	Variant type	Inheritance	Genetic pattern	ACMG	DS	Mutation Taster	PolyPhen-2	PROVEAN	Functional Prediction	Reported
1	c.4361 A > G	p.Glu1454Gly	Missense	De novo	AD	P	Yes	Disease causing (0.99)	Probably damaging(1)	Deleterious(-6.187)	LOF	No
2	c.1178G > C	p.Arg393Pro	Missense	De novo	AD	P	Yes	Disease causing (0.99)	Probably damaging(1)	Deleterious(-6.875)	LOF	Yes
3	c.2131 C > T	p.Gln711Ter,1299	Nonsense	De novo	AD	P	Yes	-	-	-	-	Yes
4	c.3800T > C	p.Met1267Thr	Missense	Paternal	AD	LP	No	Disease causing (0.99)	Probably damaging(1)	Deleterious(-5.661)	LOF	No
5	c.4339- 1(IVS22) G > A	—	Splicing	De novo	AD	P	Yes	-	-	-	-	Yes
6	c.2575 C > T	p.Arg859Cys	Missense	De novo	AD	P	No	Disease causing (0.99)	Probably damaging(1)	Deleterious(-7.346)	LOF	Yes
7	c.4268T > C	p. Leu1423Pro	Missense	Unknown	AD	LP	No	Disease causing (0.99)	Probably damaging(1)	Deleterious(-6.672)	LOF	No
8	c.5522T > C	p.Leu1841Pro	Missense	De novo	AD	LP	No	Disease causing (0.99)	Probably damaging(0.913)	Deleterious(-7.348)	LOF	Yes
9	c.5020G > C	p.Gly1674Arg	Missense	De novo	AD	P	No	Disease causing (0.99)	Probably damaging(1)	Deleterious(-6.963)	LOF	Yes
10	c.631 A > G	p.Asn211Asp	Missense	De novo	AD	P	No	Disease causing (0.99)	Possibly damaging(0.743)	Deleterious(-4.739)	GOF	No
11	c.5641G > A	p.Glu1881Lys	Missense	De novo	AD	P	No	Disease causing (0.99)	Probably damaging(0.995)	Deleterious(-3.648)	No functional effect	Yes
12	c.3429(exon18)_c.3429 + 1(IVS18)insCTT	p.Lys1144insLeu	Insertion	De novo	AD	LP	No	-	-	-	-	No
13	c.1702 C > T	p.Arg568Ter,1442	Nonsense	De novo	AD	P	Yes	-	-	-	-	Yes
14	c.3733 C > T	p.Arg1245Ter,765	Nonsense	De novo	AD	P	Yes	-	-	-	-	Yes
15	c.724 C > G	p.Gln242Glu	Missense	Maternal	AD	LP	No	Disease causing (0.99)	Probably damaging(0.998)	Deleterious(-2.931)	GOF	No
16	c.2815_c.2816 insTTCC	p.His939Leufs*59	Frameshift	De novo	AD	P	No	-	-	-	-	No
17	c.2589 + 3(IVS14)A > T	—	Splicing	De novo	AD	P	Yes	-	-	-	-	Yes
18	c.4699delG	p.Glu1567Asnfs*3	Frameshift	De novo	AD	P	Yes	-	-	-	-	No
19	c.5115_c.5116 insC	p.Asn1706Glnfs*3	Frameshift	De novo	AD	P	No	-	-	-	-	No
20	c.5725 A > G	p.Thr1909Ala	Missense	De novo	AD	LP	No	Disease causing (0.99)	Probably damaging(1)	Deleterious(-4.602)	Unreliable	Yes
21	c.1655delC	p.Pro552fs*6	Frameshift	De novo	AD	P	No	-	-	-	-	No
22	c.3818 C > T	p.Ala1273Val	Missense	De novo	AD	P	No	Disease causing (0.99)	Probably damaging(1)	Deleterious(-3.737)	Unreliable	Yes
23	c.2435 C > G	p.Thr812Arg	Missense	Maternal	AD	LP	Yes	Disease causing (0.99)	Possibly damaging(0.703)	Deleterious(-4.944)	LOF	Yes
24	c.829T > G	p.Cys277Gly	Missense	De novo	AD	P	No	Disease causing (0.99)	Probably damaging(1)	Deleterious(-11.647)	Unreliable	Yes
25	c.5765T > C	p.Ile1922Thr	Missense	De novo	AD	P	No	Disease causing (0.99)	Probably damaging(1)	Deleterious(-4.409)	Unreliable	Yes
26	c.2134 C > T	p.Arg712Ter,1298	Nonsense	De novo	AD	P	No	-	-	-	-	Yes
27	c.1010G > A	p.Gly337Glu	Missense	De novo	AD	LP	No	Disease causing (0.99)	Probably damaging(1)	Deleterious(-7.639)	LOF	Yes
28	c.268T > G	p.Phe90Val	Missense	De novo	AD	LP	No	Disease causing (0.99)	Probably damaging(0.992)	Deleterious(-6.003)	LOF	No
29	c.5111T > C	p.Met1704Thr	Missense	De novo	AD	LP	Yes	Disease causing (0.99)	Probably damaging(0.999)	Deleterious(-5.090)	LOF	Yes
30	c.821G > C	p.Arg274Thr	Missense	Paternal	AD	VUS	No	Disease causing (0.99)	Probably damaging(0.982)	Deleterious(-5.514)	LOF	No
31	c.2378 C > T	p.Thr793Met	Missense	Paternal	AD	VUS	No	Disease causing (0.99)	Probably damaging(0.995)	Deleterious(-5.365)	LOF	Yes
32	c.4063 C > T	p.Leu1355Pro	Missense	Paternal	AD	LP	No	Disease causing (0.99)	Probably damaging(1)	Deleterious(-3.085)	LOF	Yes
33	c.2537 A > G	p.Glu846Gly	Missense	De novo	AD	LP	No	Disease causing (0.99)	Possibly damaging(0.762)	Deleterious(-6.429)	Unreliable	Yes
34	c.5354T > A	p.Ile1785Asn	Missense	De novo	AD	LP	No	Disease causing (0.99)	Probably damaging(1)	Deleterious(-6.442)	LOF	No
35	c.4906 C > T	p.Arg1636X	Nonsense	De novo	AD	P	No	-	-	-	-	No
36	c.949T > A	p.Tyr317Asn	Missense	De novo	AD	P	No	Disease causing (0.99)	Probably damaging(1)	Deleterious(-6.899)	No functional effect	No
37	c.2103G > T	p.Met701Ile	Missense	Maternal	AD	VUS	No	Disease causing (0.99)	Benign(0.008)	Neutral(-2.281)	No functional effect	No
38	c.374delT + c.365_367delTTA	p.Leu125Trpfs*9+(p.122_123delIKinsK)	Frameshift	de novo	AD	P	Yes	-	-	-	-	No
39	c.3724_3725del	p.Ile1242fs	Frameshift	De novo	AD	P	Yes	-	-	-	-	No
40	c.3880-2(IVS22)A > G	—	Splicing	De novo	AD	P	No	-	-	-	-	Yes
41	c.4958 C > A	p.Ala1653Glu	Missense	De novo	AD	P	Yes	Disease causing (0.99)	Probably damaging(1)	Deleterious(-4.355)	GOF	Yes
42	c.4510 C > T	p.Gln1504*	Nonsense	De novo	AD	P	Yes	-	-	-	-	No
43	c.2416- 1(IVS16)G > T	—	Splicing	De novo	AD	P	Yes	-	-	-	-	No
44	c.1970 C > T	p.Pro657Leu	Missense	Maternal	AD	VUS	No	Disease causing (0.99)	Benign(0.051)	Deleterious(-3.445)	Unreliable	Yes
45	c.2064_c.2074delA ATGAGAAAGA	p.Met689fs*36	Frameshift	De novo	AD	P	No	-	-	-	-	No
46	c.1121 C > T	p.Ser374Phe	Missense	Paternal	AD	LP	No	Disease causing (0.99)	Probably damaging(0.999)	Deleterious(-4.550)	LOF	Yes
47	loss1(exon:4–19)	37,575 bp	Deletion	De novo	AD	P	No	-	-	-	-	No
48	c.2729 A > G	p.Gln910Arg	Missense	De novo	AD	LP	Yes	Disease causing (0.99)	Probably damaging(0.974)	Deleterious(-3.870)	GOF	Yes
49	c.2852 A > G	p.Glu951Gly	Missense	De novo	AD	P	No	Disease causing (0.99)	Probably damaging(1)	Deleterious(-6.773)	LOF	No
50	c.2462 C > T	p.Ala821Val	Missense	Maternal	AD	VUS	No	Disease causing (0.99)	Possibly damaging(0.683)	Deleterious(-3.743)	No functional effect	No

Note: P, pathogenic; LP, likely pathogenic; VUS, uncertain significance

Variant distribution

Due to the extensive deletion at the loss1(exon4-19) variant site, statistical analysis of this deletion site was not included in the figures except for Fig. 2E. Among the 49 variant sites analyzed, the genetic variation map revealed a total of 10 cases located at exon 29 (Fig. 1A). In the quaternary structure diagram of the gene, Pathogenic variants were distributed across all regions except for the S1 and S5-S6 regions (Figs. 1B and 2A). In the SE-related statistics, no SE occurred in patients with pathological variants located in the S2-S3, S3, and S3-S4 regions, while both patients with pathological variants in the N-term region experienced SE. In the S4-S5 region, 9 cases (81.8%) did not experience SE, whereas 2 cases (18.2%) did (Fig. 2B). In the statistical analysis of EEG during the first year of onset, abnormal EEG findings were observed in children with variant loci located in the S2-S3, S3-S4, and S5 regions, while EEG results in children with loci located in the C-term showed no abnormalities. In the S4-S5 region, EEG was normal in 8 cases (72.7%) and abnormal in 3 cases (27.3%) (Fig. 2C). In the distribution statistics of variant loci based on the occurrence of DS, variant loci located in the S2-S3, S3-S4, and C-term regions were all non-DS patients, while among the variant loci in the S4-S5 region, 9 cases (81.8%) were non-DS patients and only 2 cases (18.2%) were DS patients (Fig. 2D). In the statistical analysis of pathological variant types in SCN1A, 31 cases (62.0%) were missense mutations, 7 cases (14.0%) were nonsense mutations, 6 cases (12.0%) were frameshift mutations, 4 cases (8.0%) were splice mutations, 1 case (2.0%) was an insertion mutation, and 1 case (2.0%) was a deletion mutation (Fig. 2E).

Fig. 2.

Illustrates the frequency distribution of pathological variant sites collected in this study (A-D do not include statistics for the loss1(exon4-19) variant site). Specifically, A presents the distribution of variant frequencies among pathological variant patients; B-D respectively display the distribution of variants associated with SE, whether the EEG was normal within the first year of onset, and whether it is DS syndrome. Additionally, Figure E shows the proportion of various pathological variant types

Fig. 1.

Illustrates the distribution of genetic pathological variant sites collected in this study (excluding the loss1 exon4-19) variant site). A displays the distribution of SCN1A pathological variants across the entire gene sequence; B shows the structure of the human Nav1.1 channel identified in this study and the locations of SCN1A variants, with regions marked in red indicating pathological variants associated with DS patients

Non-DS phenotypes are more prone to missense pathological variants and status epilepticus

According to the diagnostic criteria, the cases were divided into the DS group and the non-DS group. Among the 50 children, 16 were diagnosed with DS. Those located in the S2-S3, S3-S4, and C-term regions were all in the non-DS group. Among the children with variations in the S4-S5 region, 9 (81.8%) had non-DS syndrome and 2 (18.2%) had DS syndrome. The grouping into the DS and non-DS groups was based on statistical analysis of pathological variant type, whether it was a de novo variation, the presence of fever during the initial course, status epilepticus, cluster seizures, and whether the EEG was abnormal within the first year. The results showed that the P-values for missense mutations and status epilepticus were both less than 0.05 (Table 4).

Table 4.

Comparison of characteristics between the DS group and the Non-DS group

Feature		DS	Non-DS	X2	P
Missense	Yes(No)	6(10)	25(9)	5.995	0.014
Frameshift	Yes(No)	3(13)	3(31)	0.293	0.588
Splicing	Yes(No)	3(13)	1(33)	1.859	0.173
Nonsense	Yes(No)	3(14)	4(31)	0.3798	0.5377
De novo	Yes(No)	15(1)	24(9)	1.78	0.182
Fever of first seizure	Yes(No)	11(5)	21(13)	0.23	0.631
Status epilepticus	Yes(No)	9(7)	9(25)	4.188	0.041
Family history	Yes(No)	3(13)	13(21)	1.898	0.168
Cluster seizures	Yes(No)	10(6)	18(16)	0.403	0.525
Epilepsy first year EEG abnormalities onset	Yes(No)	9(7)	15(19)	0.642	0.423

Note: In this study, the total sample size was all greater than 40. When the theoretical frequency was greater than 5, the Pearson chi-square was selected. When the theoretical frequency is ≥ 1 or < 5, continuity correction is selected. When the theoretical frequency is less than 1, look at the Fisher’s exact test; P < 0.05, which was statistically significant

The relationship between the degree of neurodevelopmental disorders and the types of genetic variations

To further evaluate the impact of genotype on the severity of neurodevelopmental phenotypes, we analyzed the relationship between variant types and the grading of developmental disorders. Among the 34 children with developmental disorders, 16 (47.1%) had mild, 10 (29.4%) had moderate, and 8 (23.5%) had severe disorders. We classified nonsense, frameshift, splice site mutations, and large fragment deletions as non-missense mutations and compared them with missense mutations. The analysis revealed that the proportion of children with severe developmental disorders among those carrying non-missense mutations (7/18, 38.9%) was significantly higher than that among those carrying missense mutations (1/31, 3.2%), with a statistically significant difference (P = 0.003 < 0.05,Fisher’s exact test). All 8 children with severe developmental disorders had a history of SE, and 7 of them (87.5%) were diagnosed with DS.

Discussion

The spectrum of seizure types observed in our study further underscores the complexity of the DS phenotype. The high incidence of GTCS (48.0%) and FOS (30.0%), along with the presence of other seizure types such as MS(12.0%) and aAS (6.0%), aligns with the academic understanding of DS as an epileptic encephalopathy with multifaceted clinical manifestations [9, 10]. This heterogeneity not only increases the difficulty of early diagnosis but also emphasizes the necessity of genetic testing for children presenting with febrile seizures plus and other seizure types.This study confirms the challenges in treating SCN1A-related epilepsy: over 70.0% of the children require a combination of ≥ 2 ASMs, and although 76.0% of the children experience a ≥ 50% reduction in seizure frequency, the rate of complete seizure control is extremely low. KD, characterized by high fat, moderate protein, and low carbohydrate intake, has been demonstrated in several recent randomized controlled studies to be effective for refractory epilepsy [11–13]. In this study, All patients undergoing KD were treated with the classical ketogenic diet protocol, with a ratio of fat to combined protein and carbohydrate weight primarily at 4:1, adjusted between 3:1 and 4:1 based on individual tolerance. Notably, efficacy was observed in patients with variants p.Glu1454Gly, p.Gly1674Arg, p.Pro552fs*6, and p.Ile1922Thr. In contrast, the patient with the c.2589 + 3(IVS14)A > T splicing variant was insensitive to KD therapy. KD has demonstrated efficacy in a limited number of cases (80.0%, 4/5), while SCB may exacerbate seizures in the majority of pediatric patients (66.6%, 4/6), with only one case showing improvement and one case showing no effect.

Missense mutations (62.0%) are the predominant type of SCN1A variants. The incidence of status SE in the DS group was significantly higher than that in the non-DS group (P = 0.041), while the proportion of missense mutations was higher in the non-DS group (P = 0.014), supporting the notion that missense mutations are often associated with milder phenotypes, whereas truncating mutations tend to cause severe phenotypes. In imaging studies, MRI typically showed no specific changes, with only two cases exhibiting brain atrophy-like changes, one case showing corpus callosum dysplasia, and bilateral periventricular gray matter heterotopia. 48.0% of the children exhibited abnormal EEG within one year of onset, with background slowing being the most common manifestation, followed by spike-and-slow-wave or sharp-and-slow-wave discharges. Pathogenic variants in the S2-S3/S3-S4 transmembrane regions all resulted in abnormal EEG, which may be related to the impairment of GABAergic neuronal activity due to Nav1.1 loss of function [14–16]. Notably, children with pathogenic variants in the C-terminal and S4-S5 regions had a higher rate of normal EEG in the early stage (72.7% in the S4-S5 region), suggesting that brain electrical activity may not be significantly affected in the initial phase of the disease. Research indicates that although SCN1A pathogenic variants have caused abnormal neuronal function at the molecular and cellular levels, these early microscopic changes may be difficult to detect on EEG, suggesting that the brain network remains in a potential compensatory state. Among patients with pathogenic variants in the C-terminal region, the proportion of normal EEG findings is higher, possibly because, in the early stages, the brain network maintains normal electrical activity through compensation mechanisms that have not yet been exhausted. As the disease progresses, that is, as the network’s compensatory capacity gradually weakens, functional damage to neurons accumulates progressively, but this is merely our speculation and requires further relevant experiments to confirm, especially since DS children are more prone to developmental regression and EEG deterioration one year after onset, necessitating dynamic monitoring [17, 18]. 52% of pediatric patients exhibit normal early EEG (especially those with pathogenic variants at the C-terminal/S4-S5 region), indicating that relying solely on EEG screening is prone to missed diagnoses. Comprehensive evaluation incorporating thermal sensitivity, cluster seizures, and genetic testing is essential—for instance, although EEG may appear normal in cases of N-terminal pathogenic variants, the incidence of SE reaches 100% (2/2), necessitating heightened vigilance.

The proportion of missense variants was significantly higher in non-DS patients (P = 0.014). All missense variants predicted as “no functional impact” by FunNC (4/4) were exclusively observed in the non-DS group. Moreover, this group exhibited a more diverse spectrum of functional predictions (including LOF/GOF/no impact). Based on this data trend, we hypothesize that partial loss-of-function variants may retain some channel activity, thereby more likely resulting in milder phenotypes, whereas more severe LOF variants tend to cause DS. This inference is supported by multi-level evidence: In a large clinical cohort, Gallagher et al. systematically analyzed 1,018 pediatric cases with SCN1A variants, demonstrating a significant correlation between functional impact scores and clinical phenotype severity, with moderate-to-low impairment missense variants being more prevalent in non-DS patients [3]. At the mechanistic level, Scn1a^+/− models revealed that 50% reduction of NaV1.1 leads to impaired function in both GABAergic and glutamatergic neurons, causing excitation-inhibition imbalance, which supports the biological plausibility of “greater LOF → more severe phenotypes” [14]. It must be emphasized that our perspective is constructed based on statistical associations and literature consistency, representing a prudent mechanistic hypothesis. Further validation through future electrophysiological testing or knock-in models will be required to confirm the actual functional impacts of different missense variants. The significantly increased incidence of SE in the DS group may be related to the following mechanisms: inhibitory neuron dysfunction—loss of Nav1.1 function weakens the activity of GABAergic neurons, leading to abnormally elevated cortical excitability. Additionally, 86.0% of the children exhibited thermal sensitivity, and elevated body temperature may exacerbate the channel dysfunction caused by Nav1.1 pathogenic variant, further disrupting the excitation-inhibition balance.This study also found that the severity of neurodevelopmental disorders is significantly associated with the type of genetic variation. The risk of severe developmental disorders caused by non-missense mutations is significantly higher than that caused by missense mutations (P = 0.003), which is consistent with their loss-of-function mechanism: such mutations typically lead to complete inactivation of the allele, resulting in more profound disruption of inhibitory neuron function; whereas some missense mutations may retain residual channel function, thus presenting relatively milder phenotypes [19].

In this study, a pediatric patient (Case 10, p.Asn211Asp) was found to respond effectively to sodium channel blocker OXC treatment. Notably, the FunNC tool predicted this variant to be of GOF mechanism. This finding is highly consistent with existing literature, indicating that patients with GOF variants may respond well to sodium channel blockers, as these drugs can counteract neuronal hyperexcitability caused by GOF, which aligns with the findings of Matricardi et al., who reported that 73% of patients with GOF variant responded effectively to SCB treatment [5, 20, 21]. The expression of SCN1A in the human brain gradually increases from the neonatal period to adulthood, and SCN1A pathogenic variants can lead to the occurrence of GOF or Loss of function, indicating that different SCN1A variants may affect inhibitory or excitatory neuronal functions in distinct ways. With the deepening of research into the pathogenic mechanisms of SCN1A, SCB may potentially be applied to certain specific types of SCN1A mutants in the future [22–24]. Future research should incorporate electrophysiological experiments (such as patch-clamp techniques) to clarify the impact of different pathogenic variant types (such as missense mutations and frameshift mutations) on Nav1.1 channel function, providing a theoretical basis for precise medication. This study identified 24 previously unreported pathogenic variant sites, and the distribution of variants was correlated with SE and EEG abnormalities (such as the association between N-terminal variants and SE. Subsequent studies could expand the sample size and integrate bioinformatics prediction tools (such as Mutation Taster and PolyPhen-2) to construct a three-dimensional association model of pathogenic variant sites, functions, and phenotypes, thereby improving the accuracy of early diagnosis. Although KD is effective for 80% of pediatric patients, its specific mechanisms remain unclear. It is speculated that KD may improve the excitation-inhibition imbalance caused by SCN1A pathogenic variants by regulating mitochondrial function, enhancing GABAergic inhibition, or altering neuronal energy metabolism. Future research could further elucidate the molecular targets of KD through techniques such as metabolomics and single-cell sequencing [25–27]. Based on the discovery of GOF pathogenic variants, future exploration could focus on the precise application of SCB in specific pathogenic variant types (such as dose optimization for GOF variants) and gene editing therapies (such as CRISPR-Cas9 correction of pathogenic variants). This study, through analyzing the genotype-phenotype correlation in children with SCN1A pathogenic variants, reveals the association mechanism between missense mutations and the occurrence of non-DS, SE, and the functional impact of regional variants (such as N-terminal→SE) on EEG abnormalities. Subsequent research needs to deepen the functional validation of pathogenic variants and the study of targeted strategies to improve the prognosis of affected children.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

SE

Status epilepticus

EEG

Electroencephalogram

GEFS+

Febrile seizures plus

DS

Dravet syndrome

KD

Ketogenic diet

SCB

Sodium channel blocker

ASMs

Antiseizure medications

MRI

Magnetic resonance imaging

ILAE

International League Against Epilepsy

ACMG

American College of Medical Genetics and Genomics

PCR

Polymerase chain reaction

GOF

Gain-of-function

LOF

Loss-of-function

DQ

Developmental Quotient

sGCS

Secondary Generalized Clonic Seizure

aAS

Atypical Absence Seizure

MS

Myoclonic seizures

GTCS

Generalized tonic-clonic seizure

FOS

Focal seizure

Author contributions

M.H., X.M. and J.S. designed and performed experiments, analyzed data, and drafted the manuscript. B.W. X.W. and H.Q. assisted with data analysis and validation. G.Z. supervised the research, acquired funding, and finalized the manuscript. All authors reviewed and approved the final version.

Funding

This research was supported by the Natural Science Foundation of Jiangsu Province (Project No. BK20241732).

Data availability

The datasets used and analyzed in the current study are available in the article.

Declarations

Ethics approval and consent to participate

This study has obtained the written informed consent of the parents when the children visited the hospital. This research was approved by the Children’s Hospital Affiliated to Nanjing Medical University. All the procedures carried out in this report comply with the ethical standards of the institution and the National research council.

Consent for publication

Consent for publication was obtained from the institution and the patient’s parents.

Competing interests

All authors have declared no conflicts of interest.