Identification of novel pathogenic genes of childhood epileptic encephalopathies

Background Epileptic encephalopathy is a devastating epilepsy with etiologies largely elusive, despite whole-gene/exon sequencing of large cohorts. This study targeted the genetic causes of childhood epileptic encephalopathy, typically Lennox-Gastaut syndrome (LGS) featured by age-dependent onset and characteristic clinical manifestations. Methods Trio-based whole-exome sequencing was performed in 235 LGS cases with individualized analyses on each trio by explainable inheritance origin with stratified frequency filtration and on each gene in four aspects, and specified statistical analyses including that on compound heterozygous variants with controls of 1942 asymptomatic parents. Animal models were used to validate the roles of novel candidate genes. Results We identified three novel causative genes, including SBF1 with de novo , CELSR2 with recessive, and TENM1 with X-linked recessive variants. Significantly higher excesses of de novo SBF1 variants and biallelic CELSR2 variants, aggregated variant frequencies of SBF1 , CELSR2 , and TENM1 , and frequency of compound heterozygous CELSR2 variants in the cases were detected. Phenotype severity/outcome was correlated with the genotype of the variants in these genes. In Drosophila , knockdown of these genes showed increased seizure-like behavior and increased firing of excitatory neurons. Sbf1 knockout zebrafish showed seizure-like behavior, premature death, and increased firing of neurons. Celsr2 knockout mice showed spontaneous seizures with epileptiform discharges. Additional 42 genes were identified as novel candidate pathogenic genes with evidence of the four genetic aspects/statistics. Conclusions This study suggests SBF1 , CELSR2 , and TENM1 are pathogenic genes of LGS and highlights the implications of phenotype subclassification and individualized analyses protocol in identifying genetic causes of human diseases. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the National Natural Science Foundation of China (grant Nos. 82171439, 82271505, and 81971216), and Guangdong Basic and Applied Basic Research Foundation (grant No. 2021A1515010986). The funders had no role in study design, data collection, and analysis, or in the decision to publish or the preparation of the manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All procedures performed were in accordance with the ethical standards of the institutional committee. Ethical approval was approved by the ethics committee of the Second Affiliated Hospital of Guangzhou Medical University (approval ethics number 2020-hs-49). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The data that support the findings of this study are available from the corresponding author upon reasonable request.