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Clinical and molecular analysis of epilepsy-related genes in patients with Dravet syndrome

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Introduction
Dravet syndrome (DS) is an infrequent progressive epileptic encephalopathy with an incidence between 1:20,000 and 1:60,000. [1][2][3] It was firstly reported by Dravet in 1978, and initially called severe myoclonic epilepsy in infancy (SMEI). In 2001, the name was officially changed into DS by the International League Against Epilepsy. DS is one of the most severe types of genetic epilepsy characterized by febrile seizures and psychomotor developmental delay after seizures onset. Mutations in SCN1A gene, which encodes the voltage-gated sodium channel al-subunit, have been shown to be responsible for at least 80% of patients with DS, and 90% of these mutations arise de novo. [4] Furthermore, DS rarely involves PCDH19, STXBP1, GABRA1, GABARG2, SCN1B, and SCN2A mutations. [5][6][7][8] In addition, individuals with SCN1A mutations exhibit widely different seizure severities ranges from intractable seizures of DS, through comparatively benign febrile seizure, to completely normal family members in some cases. It has been reported that other genetic susceptibility factors may exist and act as a partner with SCN1A mutations, leading to the various clinical phenotype of patients with DS. [9,10] Genetic modifiers including SCN2A, KCNQ2, and SCN8A have been showed to influence the clinical presentation of human GEFS+ (generalized epilepsy with febrile seizures plus) associated SCN1A mutation by mouse models. [11] Furthermore, Singh et al [12] found that variants in SCN9A may act as a genetic modifier that exacerbated mild SCN1A mutation-associated GEFS+ and as a susceptibility gene for DS. However, more definitive clinical and genetic evidence is lacking. Here, the clinical and molecular characteristics of 13 DS cases were analyzed by targeted-exome sequencing to identify the novel variants in SCN1A gene among Han Chinese children and investigate the possibility of other epilepsy-related genes contributing to the variable manifestation of DS associated with SCN1A mutations. examination results, treatment, and prognosis. The study has been approved by Ethic Committee of Zhejiang University and written informed consents were obtained from all participants or their guardians (in the case of children).
2.2. Methods 2.2.1. Diagnostic criteria. According to Classification criteria of epilepsy and epilepsy syndrome (ILAE, 2010), [13] the detailed diagnostic criteria for DS were as follows: onset age within 1 year with febrile seizures; normal mental and motor development before onset, but retrogression in the second year; longstanding generalized or lateral seizures or generalized tonic-clonic seizures, often induced by fever; multi-seizure types such as myoclonic seizures, partial seizures, and atypical absence seizures shown at 1 to 4-year-old, often presented as status epilepticus; onset characterized by heat sensitivity; mental and motor retardation after onset combined with ataxia and pyramidal signs; poor therapeutic efficacy of anti-epileptic drugs.

2.2.2.
Mutational screening of epilepsy-related genes. Blood samples from subjects were collected (5 mL, EDTA anticoagulated), and FlexiGene DNA Kit (Qiagen, Germany) was used for whole genomic DNA extraction. Exon target capture and secondgeneration sequencing were used in combination to screen mutations in 308 epilepsy-related genes (Table S1, http://links. lww.com/MD/C691). Agilent SureDesign was employed to design target capture probes on target gene exons and flanking sequences (± 10 bp) (total 60,605 with 1.961 Mbp). Then, Agilent SureSelect Target Enrichment System was combined with Illumina NextSeq 500 to perform high-throughput sequencing. Data analysis included fundamental assessment of original sequences and bioinformatics evaluation. After analysis by the RTA software (real-time analysis, Illumina San Diego, California), the CASAVA software v1.8.2 (Illumina), BWA and GATK, sequencing quality was assessed, and single nucleotide variation (SNV) reports were obtained. Furthermore, ANNOVAR, PolyPhen-2, SIFT, HGMD database, dbSNP database, and 1000 Genome database were used to conduct variation annotation and analysis to confirm candidate pathogenic mutations. To identify putative pathogenic mutation, variants associated with DS were evaluated using following criteria: recorded or reported as pathogenic mutation by HGMD and PubMed databases (reported mutation); minor allele frequency <0.01 in dbSNP and genotype frequency <0.005 in 1000 Genome databases; predicted as detrimental mutation by PolyPhen-2 and SIFT; alignment by National Center for Biotechnology Information (NCBI) BLAST Link showing highly conservation in evolution. Finally, the PCR-Sanger method was used to assess probands and their family members, as well as normal controls to confirm the pathogenic mutations.

Mutational analysis of SCN1A gene in DS patients
As shown in Table 1, targeted-exome sequencing, bioinformatic analysis, and Sanger sequencing verification revealed that 11 out  Fig. 2, the variant was inherited from his mother who displayed febrile seizures in Family 8, and the  younger brother with DS phenotype also carried SCN1A c.2714C > A (p.A905D) variant. Additionally, 2 patients in our cohort were failed to detect any pathogenic mutations in 308 epilepsy-related genes.

Mutational analysis of other epilepsy-related genes in DS patients
In addition to the SCN1A variants mentioned above, putative pathogenic mutations in other epilepsy-related genes associated with DS were also evaluated (Fig. 3). In this cohort, 2 of these DS subjects with SCN1A variants were found to be in combination with the other epilepsy-related variants. Particularly, the pediatric patient from Family 1 carried SCN9A (c.2132T > C, p.L711S) and KCNQ2 (c.1491G > C, P.G497D) variants along with SCN1A (c.4233_4234insAT, p.I1412Mfs37) variant. The variant p.L711S is located at the inactivation loop connecting DI and DII of voltage-gated sodium channel NaV1.7 which encoded by SCN9A and shared same overall structural motif as NaV1.1. The resulting P.G497D amino-acid change which occurred in the C-terminal of the potassium-channel subunit Kv7.2 (encoded by KCNQ2), was located in an evolutionarily conserved region. Furthermore, the proband (boy) in Family 3 with SCN1A c.2332-1G > C variant also harbored SLC6A8 c.1151-8C > T variant, which may affect the process of precursor mRNA into mature mRNA and lead to the production of abnormal protein. In fact, neither of 2 variants was detected in the 100 unrelated population-matched controls by Sanger sequencing. The 2 missense mutations all localized at highly conserved amino acids of proteins and may cause potential structural and functional alterations. High probability of damaging effect by PolyPhen-2 and SIFT prediction of these variants further supported their pathogenic nature, and may contribute to the variable DS phenotype associated with SCN1A mutations.

Clinical and genetic characterization of SCN1A mutated patients with additional modifier variations
A genotype-phenotype correlation analysis was carried out among 2 SCN1A mutated patients carrying additional modifier variations (Table 2). In Family 1, the proband carried SCN1A variant along with SCN9A and KCNQ2 variants has an unusually favorable cognitive and behavioral development. At admission to primary school, no obvious decline in cognitive level was observed. In Family 3, the proband with SCN1A and SLC6A8 variants exhibited more serious of speech deficits, developmental delay, and intellectual deficiency. He is currently 5 years old, and has no speech and limited language comprehension. The interictal electroencephalogram (EEG) showed slow background activity and multifocal spike slow waves. The brain magnetic resonance imaging (MRI) suggested abnormal signal in ventricular trigone. Long echo time (288 ms) proton magnetic resonance spectroscopy (MRS) reveals diminished creatine signal which was acquired within frontal white matter. Furthermore, the patient's uncle from mother's side suffered seizures, mental, and motor retardation.

Discussion
Mutations in SCN1A gene have been found to be responsible for at least 80% of patients with Dravet syndrome, and 90% of these mutations arise de novo. In the present study, we detected 11 variants (including 10 de novo variants) in SCN1A gene among 11 pedigrees, accounting for about 84.6% (11/13) of DS in our cohort. Claes et al [14] reported SCN1A gene mutation resulting in DS for the first time in 2001. Marini et al [15] indicated that SCN1A mutation accounts for ∼80% of all DS cases, while mutations in other genes only account for a small part. It has been reported that a total of 1727 SCN1A gene variants have been reported, including 1448 mutations associated with the DS phenotype. Among these, 617 variants were missense mutations, and there was no hot spot mutation. [4] In this study, SCN1A c.4233_4234insAT variant from Family 1 and SCN1A c.2985T > G variant from Family 6 has been previously reported [16,17] , while the remaining 9 variants were novel. These findings expanded the mutational spectrum for the SCN1A gene in Chinese Han population with DS. A total of 11 SCN1A putative pathogenic variants were identified among 11 pedigrees including 2 frameshift mutations, 2 splice site mutations, and 7 missense mutations. SCN1A encodes the a1 subunit of the sodium channel, and is located on chromosome 2q24. The a-subunit includes 4 homologous regions with 6 transmembrane fragments (S1-S6) each. Among them, S4 is the voltage zone. S5, S6, and the ring between them is the gate zone. The SCN1A protein coding region, especially mutations in the gate zone can lead to permeability and electroconductibility changes of the sodium channel that results in increased cell excitability and neuronal discharge with tiny stimulations, causing epilepsy. The c.4233_4234insAT variant in Family 1 and c.3078delG variant in family 10 caused an altered open reading frame, resulting in a completely different translation and dysfunction of Nav1.1. Two splice site mutations (c.2332-1G>C and c.1029-3C>A) which located at the acceptor-sites or recognition sites, leading to the abnormal processing of mRNA, were identified in Family 3 and Family 7, respectively. Amino acid synthesis and arrangement are greatly influenced by such mutations, and the function of the product is significantly changed, resulting in severe clinical manifestations. The remaining 7 missense mutations in SCN1A gene, including p.L373W, p.F945L, p.P1443V, and p.E1445K (S5-S6 pore loop regions), p.A905D (S5), p.F995L (intracellular DII and DIII connecting ring), and p.L373W (intracellular DIII and DIV connecting ring). In our study, 5 missense mutations occurred in the gate zone (71.4%, 5/7), suggesting the SCN1A missense mutation associated with DS mostly located at important functional region. Our finding was consistent with previous studies by Zuberi et al [18] and Meng et al. [4] SCN1A mutations in DS pediatric patients were mostly de novo, and their clinical phenotypes were greatly different. [19] In our study, 10 of these SCN1A variants in DS patients were de novo, except the SCN1A c.2714C > A (p.A905D) variant. Sanger sequencing showed that this variant was inherited from his mother who exhibited febrile seizure before 6 years of age. In addition, his younger brother with DS phenotype also carried this variant. Several studies have reported that some DS subjects inherited SCN1A mutations from asymptomatic parents or mildly affected adult patients. Actually, somatic and germline SCN1A mosaicism has been reported in a small number of cases. [20,21] Somatic mosaicism may account for variable expressivity of SCN1A mutations in SMEI families.
Genetic interaction between sodium channel mutations and variants in other channels have been described both in mouse models and affected patients. [11] These results suggest that ion channel variants may contribute to the clinical variation observed in subjects with monogenic epilepsy. Particularly, interactions between genetic variations of SCN1A and KCNQ2 observed in the mouse and mutations of SCN1A and SCN9A described in DS patients provide important models for potential genetic modifier effects of DS. [12] In our cohort, SCN1A Table 2 Summary of clinical and molecular data for 11 DS patients with puptative pathogenic variants on epilepsy-related genes.  mutations were found to be combined with other epilepsyrelated mutations (SCN9A and KCNQ2 variants in Family 1 and SLC6A8 variant in Family 3) that may contribute to the variable phenotype of SCN1A-related DS. In Family 1, segregation analysis showed that the SCN9A p.L711S and KCNQ2 p.G497D variations were inherited from the asymptomatic father without a family history of seizures. It has been reported that SCN9A variations themselves alone might be asymptomatic or cause infrequent febrile seizures due to incomplete penetrance and variable expressivity, but likely contribute to the modification of the severity of SCN1A related DS. There is indeed that a subset of DS patients commonly with mutations in SCN1A gene also harbor SCN9A mutations. [22] In addition, KCNQ2 mutation has been considered to be as a cause of seizures benign neonatal and epileptic encephalopathy early infantile. [23] Previous study have demonstrated that KCNQ2 mutation could exhibit increased susceptibility to induced seizures, and can dramatically influence the phenotype of mice carrying the SCN1A mutation. [24] However, it is reassuring that the proband in Family 1 has undergone an unusually favorable cognitive and behavioral development in comparison with other DS subjects. Furthermore, mutations in SLC6A8 has been reported to be associated with X-linked cerebral creatine deficiency syndrome1 (CCDS1), and speech difficulty is considered as a hallmark of CCDS. [25] The defect of SLC6A8 impaired the transport of creatine into brain and resulted in creatine transporter deficiency, thereby producing the phenotype of CCDS1. The SCL6A8 c.1151-8C>T variant that identified in proband 3 has been reported to be pathogenic in X-linked recessive inherited model. [26] Clinical evaluation showed that his speech deficits, developmental delay and intellectual deficiency are more serious than other DS patients, MRS reveals diminished creatine signal. Thus, these findings suggested that SCN9A, KCNQ2, and SCL6A8 may act as modifiers influenced the phenotypic manifestation of the DS associated SCN1A mutations. Furthermore, these modifying alleles may preferentially found in DS patients with SCN1A mutations that are less deleterious when compared with complete heterozygous loss of function mutations. Additional functional data in mouse models to evaluate the interaction between the variants in genetic modifiers and SCN1A gene will be required to test if the modification is valid in certain DS subjects.
In conclusion, these data suggested that SCN1A putative pathogenic variants account for about 84.6% of DS in our cohort. The study expanded the mutational spectrum for the SCN1A gene, and also provided clinical and genetic evidence for the hypothesis that genetic modifiers may contribute to the variable manifestation of DS patients with SCN1A mutations. Thus, targeted-exome sequencing will make it possible to detect the interactions of epilepsy-related genes and reveal their modification on the severity of SCN1A-related DS. The hypothesis of genetic modifiers on SCN1A-related variable phenotype should require more evidences from clinical patients and mouse models. Additionally, the whole exome or genome screening would be helpful to explain the molecular pathogenesis of the 2 patients who failed to detect any pathogenic mutations in 308 epilepsy-related genes.