Introduction

SCN9A gene encodes the voltage-gated sodium channel NaV1.7, one of the nine known α members of voltage-gated sodium (Nav) channels [1]. It mainly expresses in dorsal root ganglion neurons, thus its mutations are mainly associated with pain disorders [2]. However, more and more studies have shown that SCN9A mutations in patients are also associated with variable epilepsy phenotypes including febrile seizures (FS) [3], GEFS+ [4], and Dravet syndrome (DS) [5] in recent years. GABRG2 gene (γ-aminobutyric acid receptor subunit gene), SCN1A gene, and GABRA1 gene are most frequently mentioned in GEFS+ [6,7,8], while the role of SCN9A gene in GEFS+ still remains unknown. Here, we report a novel previously unreported likely pathogenic SCN9A Y1958C heterozygous mutation with no SCN1A mutations in a Chinese family with GEFS+ and explore the possibility of SCN9A contributing to GEFS+.

Material and methods

Family recruitment

A family with 10 living Han members across three generation participated in the study. All the subjects signed the informed consent. Clinical data were collected from all members.

Targeted exon capture and sequencing

After signing the consent, 2~4-ml peripheral blood from the ten members were collected. Blood Genome Column Medium Extraction Kit (Kangweishiji, China) was used to extract genomic DNA from blood samples. Whole-exome enrichment was performed using IDT_xGEN, which targets 39Mb protein-coding region of the human genome and covers 51Mb of partial intron. High-throughput sequencing was performed by Illumina NOVASeq 6000 series sequencer; the sequencing process was performed by Beijing Chigene Translational Medicine Research Center.

Sequence alignment and variant calling

All reads were mapped to the human reference sequence (hg 19) using BWA-MEM (version 0.7.12). Then, a genome analysis tool kit (GATK version 3.4.0) was used to refine the alignments by performing local indel realignment and subsequent base quality recalibration. Single-nucleotide variants (SNVs) and insertions/deletions (indels) were called with the haplotype caller of the GATK. The variant was compared against publicly available databases such as the 1000 Genomes Project and the Exome Aggregation Consortium database (ExAC). At last, protein damage analysis was conducted to qualitatively predict the probability of the results by SIFT, PolyPhen-2, and MutationTaster, and multispecies alignments were performed using Mega 7.0 to determine whether the affected amino acids were conserved.

Results

Case description

The proband (IV1) is a 9-year-old boy with normal spontaneous vaginal delivery and development. He presented the first seizure at 1 year and 4 months when encountering a respiratory tract infection with a fever (41 °C). Since then, he experienced febrile seizures for 9 times in total. The eighth seizure occurred at the age of 4, and he did not have convulsions in the next 4 years. The seizure patterns of the first time to the eighth time were all presented as generalized tonic-clonic seizures (GTCS), while the patient showed GTCS and absences on the ninth seizure when he was 9 years old with a fever. All the seizures last for about 1–2 min and could remit spontaneously. No obvious abnormality was found on neurological examination and brain magnetic resonance imaging (MRI). One week later after the last seizuring, electroencephalogram monitoring showed atypical spike-and-slow waves in the right temporal regions during sleep. In the further pedigree investigation, the proband’s father (III3) experienced febrile seizures at about 1 year old and one of the proband’s aunts (III1) reported febrile seizures at the age of two (Fig. 1a). The seizure patterns were also described as GTCS. Their seizures remitted spontaneously at that time and have not recurred until now. The proband’s grandmother (II4) and grandfather (II3) did not remember whether they had convulsions in their childhood. Besides, the family history of two dead family members (I1, I2) could not be obtained. The other family members denied seizure. None of them were treated with antiepileptic drugs for their seizures.

Fig. 1
figure 1

a Family pedigree. The black arrow indicates the proband; the legend for the symbols is at the right top of the figure. b Identification of a heterozygous mutation c.5873A>G (p.Y1958C) in the family members: proband (IV1), proband’s father (III3), proband’s aunt (III1), and proband’s grandmother (II4). Additional Sanger sequencing results are given in Online Resource (Figs. 3 and 4) The red arrow shows an A to G transition of nucleotide 5873

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Genetic findings

A novel heterozygous SCN9A mutation (c.5873A>G) was detected in the proband (IV1), proband’s father (III3), proband’s aunt (III1), and proband’s grandmother (II4) by using clinical whole-exome sequencing, and Sanger sequencing was used to validate it (Fig. 1b). No mutations in SCN1A were detected. This mutation (c.5873A>G chr2:167055243 p.Y1958C) occurs in the population at a frequency of < 0.5% in the ExAC database (http://exac.broadinstitute.org/variant/2-167055243-T-C) and has not been reported in previous study or presented in dbSNP (http://evs.gs.washington.edu/EVS/) and 1000 Genomes Project (https://www.internationalgenome.org/). Multiple sequence alignment was performed by using Mega 7.0 (https://www.megasoftware.net/), and residue Y1958 is highly conserved (Fig. 2). The results of three bioinformatics programs show that the novel mutation could damage the function of the protein (Table 1). The pathogenicity of this variant is classified likely pathogenic, following the principle of standards and guidelines recommended by ACMG in recent publication [9].

Fig. 2
figure 2

Alignment of multiple SCN9A protein sequences across species. The Y1958C affected amino acid locates in the highly conserved amino acid region in different mammals (from Ensembl). Red column shows the Y1958C site

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Table 1 The prediction of the identified variant in SCN9A
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Discussion

GEFS+ is a complex autosomal dominant disorder with conspicuous phenotypic heterogeneity [10]. The first and second frequent phenotypes are febrile seizures, where generalized tonic-clonic seizures (GTCS) with fever occur between 3 months and 6 years, and febrile seizures plus (FS+), in which attacks with fever extend beyond 6 years or afebrile GTCS occur, respectively. The other phenotypes include FS/FS+ with absence, myoclonic, atonic, or focal seizures [11]. In our study, the proband experienced FS and FS+ with absence while his father and aunt only had febrile seizures, probably due to the incomplete penetrance and/or the phenotypic heterogeneity. As for the proband’s grandmother (II4), she did not remember whether convulsions had occurred in her childhood.

Several genes have been announced to be associated with GEFS+, in which GABRG2, SCN1A, and GABRA1 account for the major part [6,7,8], while SCN9A is only reported in a few cases. A potentially pathogenic SCN9A variant, L266M (in exon 7), was discovered in one GEFS+ family [5]. Cen et al. reported a small pedigree diagnosed as GEFS+ with a heterozygous mutation (Q10R) in SCN9A gene without SCN1A mutation [4]. In 2019, a heterozygous mutation in the SCN9A gene, p.(Lys655Arg), in two sisters from a non-consanguineous family who presented GEFS+ was detected [11]. Actually, SCN9A variant is often mentioned as a genetic modifier in SCN1A mutation-associated epilepsy. A heterozygous SCN9A mutation, p.N641Y, was found to be responsible for a large Utah family (K4425) suffering from FS and GEFS+, and the authors identified nine SCN1A mutations with six different SCN9A mutations in this study [3]. However, the exact role of SCN9A mutations without SCN1A variants in GEFS+ has still remained left unclear. Thus, we hope to provide more evidence to illustrate that SCN9A has an important bearing on GEFS+ development in addition to these cases.

SCN9A, encoding sodium channel Nav1.7, contains 27 exons on chromosome 2q24.3 [12]. Nav1.7 is composed of 1977 amino acids and is organized into 4 domains, each with 6 trans-membranes [1]. In our study, a novel SCN9A heterozygous mutation (c.5873A>G) causing a missense mutation (p.Y1958C) was discovered. Regrettably, the mutation didn’t occur in the 4 and the exact cellular mechanism is unclear. However, the mutation in our study was located in highly conserved positions. The SCN9A N641Y mutation in FS proved to reduce thresholds to electrically induced seizures, and increase seizure susceptibility by targeted knock-in mouse model was also in highly conserved positions [3]. So, we speculate that the SCN9A Y1958C mutation might also affect the selectivity of the ion channel. Besides, the bioinformatics programs also demonstrated that the novel mutation could damage the function of the protein. All the evidences confirmed that the SCN9A p.Y1958C mutation should be regarded as pathogenic mutation in this family.

This report further supports that SCN9A mutation without SCN1A mutations is associated with GEFS+ and expands the spectrum of SCN9A gene, but there are limitations in our study that should be addressed. Thus, the functional effect of the mutation should be further studied to strengthen our views.