Research Article
Molecular Aspects of Dravet Syndrome Patients in Taiwan
Wei-De Lina,d, Kai-Ping Changh, Chung-Hsing Wangb,f, Shyi-Jou Cheni, Pi-Chuan Fanj,
Wen-Chin Wengj, Wei-Chiang Link,l, Yushin Tsaig, Chang-Hai Tsaib, I-Ching Choub,e *, Fuu-Jen
Tsaia,b,c,g,m *
a Department of Medical Research, b Department of Pediatrics, c Department of Medical
Genetics, China Medical University Hospital, Taichung, Taiwan
d School of Post Baccalaureate Chinese Medicine, e Graduate Institute of Integrated Medicine, f School of Medicine, g School of Chinese Medicine, China Medical University, Taichung,
Taiwan
h Department of Pediatrics, Taipei Veterans General Hospital, Taipei, Taiwan i Department of Pediatrics, Tri-Service General Hospital, Taipei, Taiwan
j Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan
k Department of Biomedical Engineering, Florida International University, Miami, FL, USA l Neuro-Engineering, Miami Children's Hospital, Miami, FL, USA
m Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan
Running title: Molecular aspect of DS in Taiwan
*Correspondence author: Dr. I-Ching Chou or Dr. Fuu-Jen Tsai
Department of Pediatrics and Medical Genetics, China Medical University Hospital, No. 2, Yu-De Road, 404, Taichung, Taiwan
Tel: 886-4-22052121 ext. 2066 or 2041 Fax: 886-4-22033295
Abstract
Background: Dravet syndrome (DS) is a rare form of intractable epilepsy. Children with DS often start having seizures in infancy, and gradually develop other seizure types. Several studies have demonstrated that certain gene mutations and submicroscopic copy number variations (CNV) in DS patients are strongly associated with intractable epilepsy. In this study, directed DNA sequencing and microarray technology were used to investigate genomic variations in DS patients.
Methods: A total of nine DS patients were enrolled in this genetic study. A detailed medical history was obtained from each participant, and appropriate neurological examinations performed. Seizure types and epilepsy syndromes were classified according to ILAE criteria. The complete coding regions of SCN1A, SCN1B, SCN2A, GABRG2, and GABRD, including the intron/exon boundaries, were sequenced using DNA samples drawn from participants. In addition, whole genome CNV analysis was conducted via SNP microarray analysis.
Results: DNA sequencing revealed a mutation in the SCN1A gene in five (55.6%) of the DS patients, within which three missense mutations, c.719T>C (p.Leu240Pro), c.2807A>T (p.Asp936Val), c.4349A>C (p.Gln1450Pro), and two frameshift mutations, c.2277insAACA (p.His759fsX772) and c.3972insT (p.Leu1324fsX1331) were observed. Upon CNV analysis, a novel duplication region, 4q13.1-q13.2, was detected in one DS patient; this variant region contained a gene, EPHA5, related to cerebral neuron development.
Conclusion: This study extended the spectrum of SCN1A mutations in Taiwanese DS patients and confirms the high sensitivity of SCN1A for the DS phenotype. In addition, a novel
duplication region identified within EPHA5 should be considered in future screening procedures for DS.
1. Introduction:
Dravet syndrome (DS, MIM#607208) is a rare type of intractable epilepsy in infants. The onset of DS is usually during the first year of an infant’s life. It eventually leads to the development of other seizure types that may become status epilepticus. Since seizures in DS patients are often intractable, ataxia, slowed psychomotor development, and/or mental decline are frequently observed [1]. Currently, there is no effective treatment for DS. Consequently, their long-term prognosis usually is determined by their seizure frequency [2-4].
Several recent studies have identified strong associations between certain gene mutations and idiopathic generalized epilepsy. For example, mutations in the voltage-gated sodium channels type I-alpha subunit (SCN1A), beta subunit (SCN1B), type II-alpha subunit (SCN2A), gamma-aminobutyric acid receptor-gamma-2 (GABRG2) and gamma-aminobutyric acid receptor-delta (GABRD) result in genetic (generalized) epilepsy with febrile seizures plus (GEFS+) [5-9]. To date, several studies have suggested that mutation in the SCN1A gene is the primary cause of refractory seizures in DS patients [10-12]. In addition, Harkin reported mutation in the GABRG2 gene in a single family with DS [13]. Both of these gene mutations are spontaneously dominant mutation types [14]. Since the frequency of the SCN1A gene mutation in DS patients is between 50 to 80%, it is believed that other genes or factors are involved in DS development [15].
employed in genome-wild association studies to identify new loci associated with disease. It also can be used to detect submicroscopic copy number variations (CNV) at the whole genome level. Because of SNP microarray analysis, scientists have been able to uncover the involvement of micro-deletions or micro-duplications in many disorders [16], and to
demonstrate CNV as an important causative factor in various neurological diseases, including Tourette syndrome [17], autism spectrum disorders [18], and epileptic encephalopathies [19].
In this study, we utilized two molecular methods to investigate genomic variations in DS patients. The first method was directed DNA sequencing. Since DS is strongly associated with SCN1A gene mutations and is similar in phenotype to GEFS+, SCN1A, SCN1B, SCN2A, GABRG2 and GABRD were targeted in the sequencing analysis. The second method was SNP microarray analysis, which was used to screen CNV at the whole-genome level.
2. Materials and methods
2.1. Patients
A total of nine DS patients were enrolled in this study. Among them, seven were males and two females. Their ages were between five to ten years old. All participants underwent extensive medical examination, EEG evaluation, and brain magnetic resonance imaging (MRI). Seizure types and epilepsy syndromes were classified as to the febrile seizure category according to the ILAE criteria [20]. The characteristics include normal development before onset, seizures beginning during the first year of life in the form of generalized or unilateral febrile clonic seizures, secondary appearance of myoclonic jerks and often partial seizures. EEGs show generalized spikes and waves and polyspikes and waves, early photosensitivity and focal abnormalities. MRI studies revealed normal or partial cerebral atrophy in all participants. Psychomotor development is retarded from the second year of life and ataxia, pyramidal signs and interictal myoclonus appear. The type of epilepsy is very resistant to all form of treatment, and exacerbated by hyperthermia. Prior to genetic analysis, informed consent according to the national law was obtained from parents or the other guardians of each study subject.
2.2. DNA preparation and sequencing
Genomic DNA was extracted from each patient’s peripheral blood leukocytes using a MagNA Pure LC DNA Isolation Kit (Roche, Mannheim, Germany). Complete SCN1A, SCN1B, SCN2A, GABRG2, and GABRD coding regions, including intron/exon boundaries,
were amplified according to a procedure reported in previous literature [6,9,21-23]. PCR products from the above genomic DNA amplification procedure were purified from the agarose gel using QIAEX II (Qiagen, Hilden, Germany) and then used for direct sequencing to detect gene mutations. The direct sequencing process was performed using the BigDye 3.1 Terminator cycle sequencing kit (Applied Biosystems, Forest City, CA, U.S.A.) with the ABI 3100 Genetic Analyzer (Applied Biosystems, Forest City, CA, U.S.A.).
To determine the carrier-rate of the novel mutations detected in the Taiwanese population, the SCN1A gene profile of one hundred healthy individuals was analyzed using the same procedure mentioned above. Reference sequence and base-pair numbers of SCN1A were obtained from GenBank using the accession number NM_001165963.
2.3. Whole-genome CNV microarray analysis
Whole-genome CNV analysis was performed using the CytoSNP-12 beadchips (Illumina, Inc., San Diego, CA, U.S.A.) according to the instructions provided by the manufacturer. This array set contains nearly 300,000 markers that cover genome-wide tag SNPs and markers for known cytogenetic importance regions. After DNA amplification, fragmentation, hybridization, single-base extension, labeling and washing, the chips were scanned by Illumina BeadStation 500 GX laser (Illumina, Inc., San Diego, CA, U.S.A.) to generate intensity data for each SNP. For copy number analysis and genotype calling, the intensity data were further processed using Illumina GenomeStudio and KaryoStudio software
programs (Illumina, Inc., San Diego, CA, U.S.A.) The B allele frequency (BAF) and Log R ratio obtained from CytoSNP-12 chips were used to determine the copy number in the assayed genome. Specifically, BAF represents the proportion contributed by one SNP allele (B) to the total copy number; R is the signal intensity; and the Log R ratio is the log base 2 of the measured normalized R value for a particular SNP divided by the expected normalized R value. The red line on the Log R plot indicates a smoothing series with a 200 kb moving average window [24-26]. The length and position of each variation sequence was obtained from the NCBI human genome database (Build 37.3).
2.4. CNV validation
Confirmatory evidence for CNVs was obtained using real-time quantitative-PCR (qPCR) in conjunction with the Universal Probe Library (UPL, Roche, Mannheim, Germany). The UPL probes were selected using ProbeFinder v2.45 software (Roche, Mannheim,
Germany, http://www.roche-applied-science.com). The target genes included trans-2,3-enoyl-CoA reductase-like (TECRL), EPH receptor A5 (EPHA5), and gonadotropin-releasing
hormone receptor (GNRHR). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a reference gene. All reactions were performed in triplicate; each 96-well plate included three control samples and four genes (one reference and three targets), as well as a no-template control for each gene. The plate was analyzed with a LightCycler 480 Real-Time PCR system (Roche, Mannheim, Germany), and raw data were acquired using LightCycler
480 software. The formula used to calculate copy number was: copy number = 2 * 2-(Ctp - Ctn)
where Ct is the threshold cycle defined as the mean cycle at which the fluorescence curve reaches an arbitrary threshold; Ct is the difference between the Ct of the target gene and that of GAPDH; Ctp is the Ct of patients; and Ctn is the Ct of normal individuals.
2.5. Computational analysis
The conservation of amino acid in mutant residues was compared against other types of SCN alpha subunit, including human: SCN2A: AAG53412, SCN3A: AAK00219, SCN4A: NP_000325, SCN5A: AAK74065, SCN7(6)A: NP_002967, SCN8A: AAF35390,
SCN9A:Q15858, SCN10A:NP_006505, SCN11A: AAF17480, and SCN12A: AAF24976; chimpanzee SCN1A: XP_003309330; gorilla SCN1A: XP_004032776; rat SCN1A: NP_110502; mouse SCN1A: NP_061203; electric eel SCN: AAA79960; and Drosophila melanogaster SCNA: AAB59195. Peptide sequence alignment was performed using the program MegAlign (DNA Star Inc. Madison, WI U.S.A.) The hydrophilicity change of Asp936Val was calculated using the program Protean (DNA Star Inc. Madison, WI U.S.A.)
3. Results
3.1. Target gene directed DNA sequencing
Results of directed DNA sequencing revealed no mutations in the coding regions of SCN1B, SCN2A, GABRG2 or GABRD in any of the nine study subjects. Mutations in SCN1A, however, were detected in five participants. These five mutations could be divided into two subgroups: missense mutations, including c.719T>C (p.Leu240Pro), c.2807A>T
(p.Asp936Val), and c.4349A>C (p.Gln1450Pro); and frame-shift mutations, including c.2277insAACA (p.His759fsX772) and c.3972insT (p.Leu1324fsX1331) (Table 1). The Leu240, Asp936 and Gln1450 were highly conserved among human and other species sodium-channel alpha-subunits (Fig. 1). Regional hydrophilicity decreased when the aspartic acid at codon 936 mutated to valine (Fig. 2).
All these DNA variations were spontaneous mutations not found in the parents or other family members of participants, or in healthy individuals. In addition to these mutations, 15 polymorphisms spread across SCN1A were observed, including: 5'UTR-83T>A,
IVS2+64T>C, IVS6-21C>T, IVS7+21T>C, IVS7-68C>T, IVS8+75C>A, IVS8+112C>T, c.1212A>G(p.Val404Val), IVS9+52G>A, IVS10-47T>G, c.2292T>C(p.Val764Val), IVS13-37A>C, IVS15-41C>T, c.3196A>G(p.Thr1036Ala), and c.3474C>T(p.Asp1158Asp).
3.2. Whole-genome CNV screening
Whole-genome CNV screening revealed no large deletion or duplication was observed in any patient’s SCN1A region. However, aberrant CNV in one participant, who did not possess
any mutations in targeted coding regions, was found. This CNV was located on chromosome four, and a 3.34-Mb duplicated DNA segment spanning from 65.08 Mb to 68.43 Mb (4q13.1-q13.2) was identified (Fig. 3). This particular subject was a seven years old boy with no family history of consanguinity. Despite uneventful prenatal and perinatal histories, he developed right arm clonic convulsions and right eye blinking with fever at two months old. At the age of two years, he started developing frequent generalized tonic-clonic seizures (GTCs), with or without low grade fever. Administration of anticonvulsants (AEDs) did not stop the frequent attacks of GTCs, myoclonic jerks, or complex partial seizures, which often were triggered by fever. Eventually, the boy’s tonic-clonic seizures became status epilepticus. Profound mental retardation had been noted since the age of two, which evolved into global developmental delay. At the age of five, MRI images of this boy’s brain revealed no obvious abnormal changes in the signal intensity of brain parenchyma, including bilateral hippocampi and myelination.
3.3. CNV validation
To confirm that the structure alterations found in the sample were not artifacts of the experimental or analytic process, qPCR was performed on the above-mentioned participant, his parents and healthy controls. Consistently, one copy gain of the EPHA5 and TECRL genes located at 4q13.1-q13.2 was detected in the proband (Fig. 4A), while there was no copy number change in these two genes in the parents. We also checked the GNRHR gene, which is
located in the nearby downstream region of duplication, and no copy number changed in any sample (Fig. 4B). These results suggest that the duplication region did not extend to the GNRHR gene. The CNV of the 4q13.1-q13.2 region was validated and confirmed in this particular participant.
4. Discussion
The topological structure of SCN1A is a monomer and consists of four homologous domains (DI–DIV). Each domain has six transmembrane segments (S1–S6), among which S4 possesses a number of positively charged amino acids and represents the voltage sensor [5,21]. According to this protein structure information, the effects of the five mutations observed in this study on SCN1A activities or structure stability are postulated. The two insertion mutations, c.2277insAAC and c.3973insT, located in the DI-DII linker and DIII S4 regions respectively, cause early termination of translation, thereby producing a C-truncated SCN1A protein from one of the SCN1A alleles (p.His759fsX772 and p.Leu1324fsX1331). These truncated transcripts or proteins could lead to loss of function and rapid degradation of SCN1A proteins.
The pore loop, which is located between S5 and S6, delineates the pore of the channel [5,21]. The four pore loops construct the lining of the channel pore, and they also confer selectivity of the channel for the sodium ion [27]. Asp936, located in the DII S5-S6 pore loop and its neighborhood, is a hydrophilic region. As we calculated, when the charged aspartic acid was replaced with non-polar valine, the hydrophilicity of this region (from codon 926 to 940) became less hydrophilic. Furthermore, the side chain of valine is neutral and short. These changes would not favor local structure stability and would disrupt channel function.
the folding rate, and makes the structure more rigid [28]. Leu240 and Gln1450 are found in the DI S4-S5 loop and DIII S5-S6 loop, respectively. These loop structures usually are flexible and facilitate polypeptide chain folding. When they are mutated to proline
(p.Leu240Pro, p.Gln1450Pro), which is a more rigid residue, the folding direction of the loop is changed, which disrupts the protein’s conformation. In previously published literature, it has been reported that the glutamine at codon 1450 may mutate to arginine and lysine in DS/severe myoclonic epilepsy in infancy patients [29,30]. Furthermore, Gln1450 is an important residue, its mutation causing severe epilepsy phenotypically.
In this study, the SCN1A mutation rate in DS was 55.6% (5 of 9). The proportion has been highly variable in previous studies, ranging from 33% to 100% [11,21,31-34]. In two previous genetic studies of Chinese patients with DS, the proportion of patients with SCN1A mutation are 77.8% and 83% [36,37]. These discrepancies may be due to the sizes of the series and the use of different clinical criteria. About the mutations type in protein level, truncating protein is the major type of mutation found in SCN1A with DS [11,21,32,35,36]. According to our results, the proportion of truncating mutations among the point mutations was 40% (2 of 5), which was similar to the previous study result in Hong Kong DS patients (46.7%) [36], but lower than the other study published by the Peking group (61.2%) [35]. Our sample numbers were limit, the proportion would be reconsidered when more samples were available. In addition, missense mutations in SCN1A are also more common mutation type
caused DS in many cases [11, 34-36]. Regarding the location of all three missense mutations identified at this time were located in the S4 to S6 regions, which are critical regions in SCN1A and many DS-related mutations are present [5,11,21,31-36]. To our knowledge, these five mutations observed in this study have not yet been reported in other population.
Micro-deletions and duplications in genomes are potential causes of several diseases. High-density SNP microarray analysis can be used to accurately assess genome
rearrangements, which provides exact gene dosage effects, thereby correlating the genotype and phenotype of a disease. By SNP microarray analysis and qPCR, a novel duplication region, located at 4q13.1-q13.2, was detected in one of the participants in this study. This variant region contained four genes: TECRL, EPHA5, CENPC1 and STAP1. Previous studies have shown that EPHA5 is one of the ephrine-A transmembrane receptors and belongs to the family of receptor tyrosine kinases. EPHA5 regulates several biological processes during embryonic development, including the guidance of axon growth cones [37,38]. In a previous animal study, EPHA5 was discovered to play key roles in cerebral neuron-development, and to exhibit continued expression in many areas of adult brain. It has been suggested that EPHA5 is one of the most important factors in brain development and synaptic plasticity [39]. In an additional study assessing for any association between CNV and autism spectrum disorder, a micro-deletion was detected in the 4q13.1 region in one patient [18]. While this micro-deletion region does not correlate with any known genes, it is very close to the
duplication region identified in this study. Base upon these observations, we believe that 4q13.1-q13.2 could be a ‘hot spot’ for neurological diseases.
Although no mutation was identified in the other three participants, it does not
necessarily exclude the possibility of other mutation types in their DS-related genes, such as inversions, translocations, and mutations within the intron and promoter. To detect these abnormalities, a more elaborated experiment/ sequencing procedure would have to be performed. In conclusion, this study extended the spectrum of SCN1A mutations and identified a new mutation profile in Taiwanese DS patients. It also validated the high sensitivity of SCN1A (55.6%) for the DS phenotype. While the novel duplication within EPHA5 identified in this study may provide some benefit in DS diagnosis, its role in the pathogenesis of DS is not yet clear and requires additional studies with larger samples. With rapid advances in DNA sequencing technologies, we should soon be able to identify more DNA variations associated with DS and, hence, uncover its true pathogenesis in future studies.
Conflict of interest
None declared.
Acknowledgments
The study was supported in part by grants from the National Science Council (NSC100-2314-B-039-026), Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH100-TD-B-111-004) and China Medical University Hospital (DMR-100-057).
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Figure legends
Fig. 1. Alignment of the three missense mutations identifid in SCN1A with other types of
human SCN alpha subunits. Highly conserved residues Leu240, Asp936 and Gln1450 were boxed. GenBank accession numbers of these proteins are: human SCN1A:
NM_001165963, SCN2A: AAG53412, SCN3A: AAK00219, SCN4A: NP_000325, SCN5A: AAK74065, SCN7(6)A: NP_002967, SCN8A: AAF35390, SCN9A: Q15858, SCN10A: NP_006505, SCN11A: AAF17480, SCN12A: AAF24976; chimpanzee SCN1A: XP_003309330; gorilla SCN1A: XP_004032776; rat SCN1A: NP_110502; mouse
SCN1A: NP_061203; electric eel SCN: AAA79960; and Drosophila melanogaster SCNA: AAB59195..
Fig. 2. Hydrophilicity plot in the neighborhood of Asp936 and mutant Val936. Relative to the
normal residue, the hydrophilic level was decreased in the mutant region.
Fig. 3. Whole-genome copy number variation scanning by CytoSNP-12 array. Data analysis
indicated novel 3.34 Mb duplication spanning from 4q13.1 to 4q13.2 (box labeled). This region includes two known genes: trans-2,3-enoyl-CoA reductase-like (TECRL) and EPH receptor A5 (EPHA5). B allele frequency: the proportion contributed by one SNP allele (B) to the total copy number. Log R ratio: the log (base 2) ratio of the normalized R value for the particular SNP, divided by the expected normalized R value. The red line on the log R
plot indicates a smoothing series with a 200 kb moving average window.
Fig. 4. Analysis of the 4q13.1-4q13.2 duplication region using real-time quantitative PCR.
(A) The copy number of the TECRL and EPHA5 genes, which were located in the
predicted duplication region, was detected in three healthy individuals (C1 to C3) and the trio. Results showed one copy gained in the proband, but not in the parents or healthy individuals. (B) The copy number of the GNRHR gene, which was located within the nearby downstream region of duplication, was detected in three healthy individuals (C1 to C3) and the trio. No copy number change was detected in any sample, implying that the GNRHR gene was excluded from the duplication region.
Table 1
Summary of mutations in the SCN1A gene observed in this study
Location in Gene Nucleotide Change a Amino Acid Change Position in Protein Structure b Mutation
Exon 6 c.719T>C p.Leu240Pro DI S4-S5 loop Missense mutation
Exon 13 c.2277insAACA p.His759fsX772 DI-DII linker Frameshift, premature stop codon
Exon 15 c.2807A>T p.Asp936Val DII S5-S6 loop Missense mutation
Exon 20 c.3972insT p.Leu1324fsX1331 DIII S4 Frameshift, premature stop codon
Exon 23 c.4349A>C p.Gln1450Pro DIII S5-S6 loop Missense mutation
a Base-pair numbers used refer to GenBank accession numbers NM_001165963. The A in the ATG of the initiator Met codon is denoted
nucleotide +1.
b The topological structure of the protein is designated by Escayg et al. [5] and Claes et al. [21]. D represents the homologous domain I to IV. S
