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Department of Neurology, the Affiliated Hospital of Putian University, Putian 351100, Fujian Province, ChinaDepartment of Neurology, Institute of Neuroscience, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, China
Department of Neurology, Institute of Neuroscience, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, China
Department of Neurology, Institute of Neuroscience, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, China
Corresponding author at: Institute of Neuroscience and Department of Neurology, the Second Affiliated Hospital of Guangzhou Medical University, Chang-Gang-Dong Road 250, Guangzhou 510260, Guangdong Province, China.
Department of Neurology, Institute of Neuroscience, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, China
We identified one putatively causative and six possibly causative variants in 60 cases with IGEs.
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GABRB1 is probably a novel causative gene for JME, which warrants recurrent support and functional studies.
•
A comprehensive evaluation combined with the ACMG scoring and assessment of clinical concordance is suggested for the pathogenicity of variants.
Abstract
Purpose
Idiopathic generalized epilepsies (IGEs) are a common group of genetic generalized epilepsies with high genetic heterogeneity and complex inheritance. However, the genetic basis is still largely unknown. This study aimed to explore the genetic etiologies in IGEs.
Methods
Trio-based whole-exome sequencing was performed in 60 cases with IGEs. The pathogenicity of candidate genetic variants was evaluated by the criteria of the American College of Medical Genetics and Genomics (ACMG), and the clinical causality was assessed by concordance between the observed phenotype and the reported phenotype.
Results
Seven candidate variants were detected in seven unrelated cases with IGE (11.7%, 7/60). According to ACMG, a de novo SLC2A1 (c.376C>T/p.Arg126Cys) variant identified in childhood absence epilepsy was evaluated as pathogenic with clinical concordance. Six variants were assessed to be uncertain significance by ACMG, but then considered causative after evaluation of clinical concordance. These variants included CLCN4 hemizygous variant (c.2044G>A/p.Glu682Lys) and IQSEC2 heterozygous variant (c.4315C>T/p.Pro1439Ser) in juvenile absence epilepsy, EFHC1 variant (c.1504C>T/p.Arg502Trp) and CACNA1H (c.589G>T/p.Ala197Ser) both with incomplete penetrance in juvenile myoclonic epilepsy, and GRIN2A variant (c.2011C>G/p.Gln671Glu) and GABRB1 variant (c.1075G>A/p.Val359Ile) both co-segregated with juvenile myoclonic epilepsy. Among them, GABRB1 was for the first time identified as potential novel causative gene for IGE.
Significance
Considering the genetic heterogeneity and complex inheritance of IGEs, a comprehensive evaluation combined the ACMG scoring and assessment of clinical concordance is suggested for the pathogenicity analysis of variants identified in clinical screening. GABRB1 is probably a novel causative gene for IGE, which warrants further studies.
]. It is characterized by generalized epileptic seizures, including absence, myoclonic, generalized tonic-clonic, and myoclonic-tonic-clonic seizures, generalized spike/polyspike-slow waves on EEG, and good prognosis with normal neurodevelopment. Generally, the IGEs comprise four syndromes, including childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE), juvenile myoclonic epilepsy (JME), and epilepsy with generalized tonic-clonic seizures alone (GTCA) [
]. Several genes have been identified in IGEs, including CACNA1H, CACNB4, CASR, EFHC1, GABRA1, GABRB3, GABRG2, HCN2, ICK, KCNMA1, SLC2A1, and SLC12A5 (OMIM, https://www.omim.org/), accounting for a small proportion (2–8%) of IGEs [
]. With the application of whole-exome sequencing, a growing number of genes and variants have been identified in patients with IGEs. However, evaluating the pathogenicity of sequence variants is often challenging, particularly in persons with variants labelled “uncertain significance”. This scenario highlights the need for a comprehensive interpretation of variants in clinical practice.
In this study, we performed trio-based whole-exome sequencing (WES) in a Chinese cohort of 60 cases with IGEs. To comprehensive interpretation of the sequence variants, the pathogenicity of variants was firstly evaluated by the American College of Medical Genetics and Genomics (ACMG) standards and guidelines, and then for a given case, the clinical concordance was assessed between the presenting phenotype of the person and the previously reported phenotype of the mutated genes.
2. Subjects and methods
2.1 Subjects
A total of 60 cases with IGEs were enrolled in the outpatient clinics of three hospitals, including the Affiliated Hospital of Putian University, the First Affiliated Hospital of Jinan University, and the Second Affiliated Hospital of Guangzhou Medical University, between June 2014 and June 2020. The clinical data were collected, including age, gender, seizure onset age, seizure types and frequency, response to anti-seizure medications (ASMs), neurodevelopment, family history, long-term video EEG, and brain MRI/CT. The procedures of open-close eyes test, intermittent photic stimulation, hyperventilation, and sleeping recording were all performed during EEG recording. Brain MRI or CT scans were performed to detect structural abnormalities. Epileptic seizures and epilepsy syndromes were diagnosed according to the criteria of the Commission on Classification and Terminology of the International League Against Epilepsy (1989, 2010, 2017, and 2022). In brief, the inclusion criteria for a case with IGE included: 1) one or a combination of seizure types restricted to absence, myoclonic, generalized tonic-clonic, and myoclonic-tonic-clonic seizures; 2) 2.5∼5.5 Hz generalized spike-wave and/or polyspike-wave discharges with normal background on EEG; 3) no or mild intellectual disability. Cases with acquired causes, such as infection, tumor, and traumatic brain damage, were excluded.
This study abided by the guidelines of the International Committee of Medical Journal Editors regarding consent with research or participation, and received approval from the Ethics Committee of the hospital.
2.2 WES and pathogenicity evaluation
Genomic DNA was extracted from the peripheral vein blood of probands and their biological parents (trios) using the Qiagen Flexi Gene DNA kit (Qiagen, Hilden, Germany). Trio-based whole-exome sequencing (WES) was conducted on Illumina HiSeq 2500 systems by BGI-Shenzhen (Shenzhen, China). Library construction, exome capture, and data processing were performed as previously reported [
]. Variants sorting and filtration were performed with WES data. First, common variants were filtered out with a minor allele frequency (MAF) ≥ 0.005 in the 1000 Genomes Project and the Genome Aggregation Database (gnomAD). Second, potential pathogenic variants were reserved, including nonsense, frameshift, canonical splice site, indels, initiation codon, and missense variants predicted to be damaging by 23 in silico tools (http://varcards.biols.ac.cn/). Third, potential disease-causing variants were screened under five models, i.e., epilepsy-associated gene [
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
], which was classified as pathogenic, likely pathogenic, uncertain significance, likely benign, and benign. Meanwhile, to assess the causality of candidate variants clinically as previously described [
], we further analyzed the observed phenotype of a given individual and evaluated the concordance with previously reported phenotypes of the candidate gene. All the candidate variants were validated by Sanger sequencing.
3. Results
3.1 General demographics and phenotypes of the IGE cohort
We recruited 60 cases with IGEs, among whom 11 were diagnosed as CAE, 15 as JAE, 31 as JME, and three as GTCA (Table 1). Fifty-five percent of the cases were males (33/60). The average ages at onset of CAE, JAE, JME, and GTCA were 6.7, 12.1, 13.3, and 15.0 years old, respectively. None of the IGE cases had intellectual disability. All IGE cases showed good responsiveness to ASMs, and 46 (76.7%) got seizure-free for more than two years.
Table 1Clinical characteristics of patients with idiopathic generalized epilepsies.
3.2 Overall yield of clinical WES analysis in the IGE cohort
In total, seven candidate variants were detected in seven unrelated cases, accounting for 11.7% (7/60) of cases with IGE. The detection rates in CAE, JAE, JME, and GTCA were 9.1%, 13.3%, 12.9%, and 0, respectively (Table 2). Three of the seven detected genes, SLC2A1, EFHC1, and CACNA1H, were established IGE-associated genes listed in OMIM. Combining the pathogenicity evaluated by the ACMG standard and assessment of clinical concordance, one gene variant was finally evaluated as putatively causative, and the other six were considered as possibly causative (Table 2).
Table 2Genes identified in idiopathic generalized epilepsies screened by trio-based whole-exome sequencing.
Phenotype
Case no.
Candidate mutation no. (%)
Putatively causative gene
Possibly causative gene
CAE
11
1 (9.1)
SLC2A1
/
JAE
15
2 (13.3)
/
CLCN4, IQSEC2
JME
31
4 (12.9)
/
EFHC1, CACNA1H, GABRB1, GRIN2A
GTCA
3
0 (0.0)
/
/
Total
60
7 (11.7)
/
/
Underline, X-linked gene.
Bold, idiopathic generalized epilepsy-associated gene listed in Online Mendelian Inheritance in Man (OMIM, https://www.omim.org/).
The clinical manifestations of the seven cases with candidate variants were demonstrated in Table 3, and their genetic features were shown in Fig. 1. The seven cases, including one with CAE, two with JAE, and four with JME, all had a typical onset age, generalized seizures with typically generalized discharges on EEG, and a good prognosis after ASMs treatments. Two JME cases had one of their parents also suffering from JME or unclassified generalized epilepsy (Fig. 1).
Table 3Clinical and genetic features of the patients with mutations.
Fig. 1Genetic data of cases with variants. (A) Pedigrees of seven cases with genetic variants and their corresponding phenotypes. m, mutant allele; +, wild type allele; Y, Y chromosome; NA, not available; *X-linked gene; red, childhood absence epilepsy (CAE); yellow, juvenile absence epilepsy (JAE); green, juvenile myoclonic epilepsy (JME); blue, idiopathic generalized epilepsy (IGE). Arrows indicate the probands. (B) Sanger DNA sequencing chromatograms in trios. The mutation sites are marked in red boxes. (C) The amino acid sequence alignments of the seven variants showed that all variants were highly conserved across vertebrates.
3.3 Pathogenicity analysis of the candidate variants
All seven candidate variants were missense variants, among which one was de novo, and the others were inherited (Fig. 1). According to ACMG, the de novo variant was evaluated as pathogenic, and the rest six inherited variants were estimated to be uncertain significance. When assessing the causality of candidate variants clinically, the clinical manifestation of each individual was consistent with the previously reported phenotype of the candidate gene, showing the variants were causative/possible causative clinically (Tables 1 and 4).
Table 4Bioinformatics analysis and ACMG scoring of mutations identified in idiopathic generalized epilepsies.
Number of algorithms predicted to be deleterious: total in silico algorithms, which was retrieved from the website http://varcards.biols.ac.cn/. Due to space limitations, only three typical results (SIFT, PP2-var, and MT) were indicated in this table.
mutation has been previously reported in references [12,14].
Path: PS1+PS2+PS3+PM1+PM2+PP3
Yes
2
JAE
CLCN4
c.2044G>A/p.Glu682Lys
0
0
0
11:21
T
B
D
MRXSRC (XL)
Novel
US: PM2+PP3
Yes
3
JAE
IQSEC2
c.4315C>T/p.Pro1439Ser
0
0
0
9:21
D
P
D
XLID1 (XL)
Novel
US: PM2+PP3
Yes
4
JME
EFHC1
c.1504C>T/p.Arg502Trp
0
0
0
16:23
D
Pr
D
JAE, JME (AD)
Novel
US: PM2+PP3
Yes
5
JME
CACNA1H
c.589G>T/p.Ala197Ser
0
0
0
19:23
T
Pr
D
CAE, EIG6 (AD)
Novel
US: PM2+PP3
Yes
6
JME
GRIN2A
c.2011C>G/p.Gln671Glu
0
0
0
19:23
D
Pr
D
FESD (AD)
Novel
US: PM2+PP1+PP3
Yes
7
JME
GABRB1
c.1075G>A/p.Val359Ile
0
0
0
13:23
T
Pr
D
DEE (AD)
Novel
US: PM2+PP1+PP3
Yes
ACMG, the American College of Medical Genetics and Genomics; AD, autosomal dominant; CAE, childhood absence epilepsy; DEE, developmental and epileptic encephalopathy; EAS, East Asian; FESD, focal epilepsy with speech disorder and with or without impaired intellectual development; gnomAD, the Genome Aggregation Database; IGE, idiopathic generalized epilepsy; JAE, juvenile absence epilepsy; JME, juvenile myoclonic epilepsy; KG, 1000 Genomes Project; MAF, minor allele frequency; MT, Mutation Taster (D, disease-causing); Path, pathogenic; PP2-var, PolyPhen-2 HVAR (B, benign; P, possibly damaging; Pr, probably damaging); SIFT, Sorts Intolerant From Tolerant (D, damaging; T, tolerated); US, uncertain significance; XL, X-linked inheritance. EIG12, idiopathic generalized epilepsy-12; MRXSRC, Raynaud-Claes syndrome; XLID1, X-linked intellectual developmental disorder-1; EIG6, idiopathic generalized epilepsy-6.
a Number of algorithms predicted to be deleterious: total in silico algorithms, which was retrieved from the website http://varcards.biols.ac.cn/. Due to space limitations, only three typical results (SIFT, PP2-var, and MT) were indicated in this table.
b Whether the clinical features of the patient were in concordance with the reported phenotype of the causative gene.
c mutation has been previously reported in references
A de novo variant in SLC2A1 (c.376C>T/p.Arg126Cys) was identified in a case with CAE. SLC2A1 has been reported to be a causative gene for IGE (EIG12, OMIM #614847). The amino acid residue Arg126 is a “hot spot” for mutations with different amino acid substitutes by cysteine, histidine, and leucine in different patients with GLUT1 deficiency syndrome or early-onset absence epilepsy [
]. The variant was evaluated as pathogenic with the ACMG standards (Table 4).
3.3.2 Inherited variants
A hemizygous missense variant of CLCN4 (c.2044G>A/p.Glu682Lys) was identified in a male patient with JAE, which was inherited from his asymptomatic mother. CLCN4 has been reported to be associated with X-linked mental retardation Raynaud-Claes syndrome (MRXSRC, OMIM #300114). CLCN4-related epilepsy comprises a broad spectrum of phenotypes ranging from mild, brief absence seizures to severe epileptic encephalopathy [
De novo and inherited mutations in the X-linked gene CLCN4 are associated with syndromic intellectual disability and behavior and seizure disorders in males and females.
De novo and inherited mutations in the X-linked gene CLCN4 are associated with syndromic intellectual disability and behavior and seizure disorders in males and females.
]. We, therefore, considered this hemizygous variant Glu682Lys to be possibly causative for the patient with JAE.
A heterozygous missense variant of IQSEC2 (c.4315C>T/p.Pro1439Ser) was identified in a female JAE patient, which was inherited from her unaffected mother with incomplete penetrance. Hemizygous and heterozygous variants in IQSEC2 have been both reported to cause X-linked intellectual developmental disorder (XLID1, OMIM #309530) with frequent seizures in both males and females [
]. Generalized epilepsy, especially absences and generalized tonic-clonic seizures, had been observed in several female patients with heterozygous IQSEC2 variants [
]. The missense variant Pro1439Ser, which was not present in general populations and was predicted to be damaging by multiple in silico tools, was considered as possibly causative from the clinical concordance.
Four heterozygous missense variants, including c.1504C>T/p.Arg502Trp in EFHC1, c.589G>T/p.Ala197Ser in CACNA1H, c.2011C>G/p.Gln671Glu in GRIN2A, and c.1075G>A/p.Val359Ile in GABRB1, were detected in four patients with JME. The variant Arg502Trp in EFHC1 and Ala197Ser in CACNA1H were inherited from their unaffected parents, showing incomplete penetrance. EFHC1 is a well-established causative gene for JME (OMIM #254770) with a penetrance of 65–78% [
]. Therefore, the two rare variants Arg502Trp and Ala197Ser were evaluated as possibly causative.
GRIN2A mutations were previously associated with focal epilepsy and speech disorder (FESD, OMIM #245570). Recently, several heterozygous missense mutations were reported in patients with IGE [
]. In this study, the missense variant Gln671Glu in GRIN2A was identified in a JME patient and her affected mother with JME (Fig. 1), showing co-segregation with JME. The variant Gln671Glu was considered possibly causative.
GABRB1 mutations were associated with developmental and epileptic encephalopathy (DEE, OMIM #617153). The patient with Val359Ile presented mild JME with an excellent prognosis, and the variant was inherited from his affected father, who also had mild IGE (co-segregation, Fig. 1). The missense variant was not present in general populations, was predicted to be deleterious by multiple in silico tools, and was highly conserved in various species (Fig. 1). Thus, Val359Ile in GABRB1 was assessed as possibly causative clinically.
4. Discussion
In the present study, we identified one putatively causative and six possibly causative variants in 60 cases with IGEs. The seven variants were found in seven genes, including three established IGE-associated genes, three genes linked to IGE, and one novel potential IGE-related gene, reiterating the genetic heterogeneity of IGEs. The variants with “uncertain significance” by ACMG were re-evaluated as possibly causative by evaluating their clinical concordance, highlighting the significance of comprehensive evaluation of pathogenicity of variants based on both ACMG scoring and clinical concordance assessment.
CAE is currently associated with ion channel genes predominantly, such as calcium channel (CACNA1H, CACNG3), GABA receptor (GABRA1, GABRB3, and GABRG2), glutamate receptor (GRM4), acetylcholine receptor (CHRNA4), and chloride channel (CLCN2) [
]. In this study, a de novo SLC2A1 missense variant was identified in a CAE patient with an onset age of four years. SLC2A1, belonging to the solute carrier family, encodes the glucose transporter that can pass through the blood-brain barrier for glucose to enter the brain. Over 350 mutations had been identified in SLC2A1, of which the majority were associated with glucose transporter type 1 deficiency syndrome (https://www.hgmd.cf.ac.uk/ac/index.php, version: HGMD Professional 2022.2). Other SLC2A1-associated phenotypes include paroxysmal exercise-induced dyskinesia and epilepsy, paroxysmal choreoathetosis with spasticity, CAE, myoclonic-astatic epilepsy, and so on. A previous study had suggested that heterozygous destructive mutations were associated with severe metabolic encephalopathy, while mild functional deficiency resulted in mild paroxysmal events [
], genetic testing should be considered for patients with early-onset absence epilepsy, especially those with movement disorders or learning disabilities. Additionally, attention should be paid to the specific treatment with ketogenic diet in individuals with SLC2A1 variants. Previous studies have demonstrated that seizures in cases with SLC2A1 variants tended to be intractable to pharmacological treatments, but responded rapidly to a ketogenic diet in 79% of cases [
], especially in cases with GLUT1 deficiency syndrome. The correct genetic diagnosis with SLC2A1 variants would be help for clinical management.
JAE shares some common genes with CAE. Previous studies showed that patients with variants in CACNB4, EFHC1, GABRA1, GRIK1, and INHA were susceptible to JAE [
]. In the present study, we identified a hemizygous CLCN4 variant in a male JAE patient and a heterozygous IQSEC2 variant in a female JAE patient, both of which were inherited from unaffected mothers. CLCN4 encodes a voltage-dependent chloride/hydrogen exchanger, which is involved in the ion homeostasis of endosomes and intracellular trafficking. IQSEC2 encodes a guanine nucleotide exchange factor that activates small GTPases and plays a critical role in the excitatory synaptic transmission. The two genes are both X-linked genes associated with intellectual disability and epilepsy [
De novo and inherited mutations in the X-linked gene CLCN4 are associated with syndromic intellectual disability and behavior and seizure disorders in males and females.
De novo and inherited mutations in the X-linked gene CLCN4 are associated with syndromic intellectual disability and behavior and seizure disorders in males and females.
]. Affected male patients usually had a more severe phenotype than female patients. Almost complete penetrance was observed in males with hemizygous mutations in both genes [
De novo and inherited mutations in the X-linked gene CLCN4 are associated with syndromic intellectual disability and behavior and seizure disorders in males and females.
]. The present study strengthened absence epilepsy as a mild phenotype in patients with CLCN4 and IQSEC2 variants.
JME is the most common IGE syndrome. EFHC1, GABRA1, CACNB4, GABRD, CLCN2, and ICK genes have been listed as JME-associated genes in OMIM (https://www.ncbi.nlm.nih.gov/omim). In this study, variants in EFHC1, CACNA1H, GRIN2A, and GABRB1 were identified in patients with JME. EFHC1 encodes the EF-hand-containing calcium-binding protein, which is involved in signaling at the synapse. It is a putative gene for JME, accounting for 3–9% of all JME cases [
]. GRIN2A encodes an N-methyl-d-aspartate (NMDA) receptor subunit that facilitates synaptic transmission and signal conduction. GRIN2A variants were found to be mainly associated with idiopathic focal epilepsy previously [
GABRB1 is probably a novel causative gene for JME, suggested by the novel GABRB1 variant co-segregated with JME identified in the present study. GABRB1, expressed predominantly in the developing brain, encodes β1 subunit of the gamma-aminobutyric acid (GABA) A receptor, a heteromeric pentameric GABA-gated chloride channel that mediate the fast inhibitory synaptic transmission in the brain. Generally, GABRB1 is predicted to have low tolerance to variation with the probability of loss-of-function intolerance (pLI) score = 0.98 (http://gnomad-sg.org/). Gabrb1-missense variants knock-in mutant mice exhibited abnormal behavior, and HEK293 cells with missense variants caused spontaneous GABA ion channel opening and increased GABA sensitivity of recombinant GABA-A receptor [
]. In humans, the β1 subunit encoded by GABRB1 displays high amino acid sequence homology (70–80%) with the β2 subunit encoded by GABRB2 and the β3 subunit encoded by GABRB3 [
]. GABRB2 and GABRB3 have been demonstrated to be associated with a broad spectrum of epilepsy syndromes with different severity, including CAE, JAE, other generalized epilepsies, and severe epileptic encephalopathies [
]. Currently, only ten GABRB1 variants, including nine missense variants and one deletion, have been registered in the HGMD database (https://www.hgmd.cf.ac.uk/ac/index.php, version: HGMD Professional 2022.2). Eight of the ten GABRB1 variants were previously reported in epilepsies [
Pupavac M, Watkins D, Petrella F, et al. Inborn Error of Cobalamin Metabolism Associated M.
Watkins D.
Petrella F.
et al.
Pupavac M, Watkins D, Petrella F, et al. Inborn Error of Cobalamin Metabolism Associated with the Intracellular Accumulation of Transcobalamin Bound Cobalamin and Mutations in ZNF143, Which Codes for a Transcriptional Activator.
]. In the present study, a novel GABRB1 missense variant was identified in a patient with JME, which was inherited from his father, who also had mild IGE. To our knowledge, this is the first report on mild generalized epilepsy with a GABRB1 variant. Clinical replication studies and experimental evidence are further needed to establish the association between GABRB1 variants and IGEs. The phenotypic spectrum of epilepsy with varied severity is also suggested for GABRB1, like its family members, GABRB2 and GABRB3.
For GTCA, the genetic etiology is highly elusive as no genetic variants have been found linked with GTCA [
]. In the present study, no variant was identified in three cases with GTCA. The sample was too small. Large sample sizes might benefit the high genetic heterogeneity of the syndrome.
There are several limitations in the study. First, the pathogenicity of these variants warrants further validation by functional studies. Second, the sample size is limited. Large cohorts are needed to explore the genotype-phenotype association in each IGE syndrome.
In conclusion, this study identified seven variants of seven causative genes in 11.7% of cases with IGE in the present cohort. Variants with de novo, co-segregated, or dominant inheritance with incomplete penetrance proved the complex trait of IGE. GABRB1 is probably a novel causative gene for JME, which warrants recurrent support and functional studies. Given the complex inheritance of IGE, a comprehensive evaluation combined with the ACMG scoring and assessment of clinical concordance is suggested for the pathogenicity analysis of variants.
Author contributions
Zhi-Jian Lin, Heng Meng, and Na He contributed to the study conception and design. Material preparation, data collection and analysis were performed by Zhi-Jian Lin, Bin Li, Peng-Xing Lin, Wang Song, and Li-Min Yan. The first draft of the manuscript was written by Zhi-Jian Lin, Bin Li, and Na He, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by the Fujian Provincial Health Technology Project (grant number 2019-ZQN-94); the Natural Science Foundation of Fujian Province (grant number 2020J011257); Science and Technology Projects in Guangzhou (33121030 and 33121173), Clinical Frontier Technology Program of the First Affiliated Hospital of Jinan University (JNU1AF-CFTP-2022-a01205); National Natural Science Foundation of China (Grant No. 81971216), Guangdong Basic and Applied Basic Research Foundation (Grant No. 2020A1515011048), Guangzhou Medical University Grant (No. 010G271099). The funders had no role in study design, data collection, data analysis, data interpretation, and decision to prepare or publish the manuscript.
Declarations of Competing Interest
None of the authors has any conflict of interest to disclose.
Acknowledgments
We thank the patients and their parents for their cooperation in this study.
References
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Latour P.
Epidemiology of idiopathic generalized epilepsies.
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
De novo and inherited mutations in the X-linked gene CLCN4 are associated with syndromic intellectual disability and behavior and seizure disorders in males and females.
Pupavac M, Watkins D, Petrella F, et al. Inborn Error of Cobalamin Metabolism Associated M.
Watkins D.
Petrella F.
et al.
Pupavac M, Watkins D, Petrella F, et al. Inborn Error of Cobalamin Metabolism Associated with the Intracellular Accumulation of Transcobalamin Bound Cobalamin and Mutations in ZNF143, Which Codes for a Transcriptional Activator.
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