YWHAZ encodes an adapter protein 14–3-3ζ, which is involved in many signaling pathways that control cellular proliferation, migration, and differentiation. It has not been definitely correlated to any phenotype in OMIM. To investigate the role of YWHAZ gene in intellectual disability and global developmental delay, we conducted whole-exon sequencing in all of available members from a large three-generation family and we discovered that a novel variant of the YWHAZ gene was associated with intellectual disability and global developmental delay. This variant is a missense mutation of YWHAZ, p.Lys49Asn/c.147A > T, which was found in all affected members but not found in other unaffected members. We also conducted computational modeling and knockdown/knockin with Drosophila to confirm the role of the YWHAZ variant in intellectual disability. Computational modeling showed that the binding energy was increased in the mutated protein combining with the ligand indicating that the c147A > T variation was a loss-of-function variant. Cognitive defects and mushroom body morphological abnormalities were observed in YWHAZ c.147A > T knockin flies. The YWHAZ knockdown flies also manifested serious cognitive defects with hyperactivity behaviors, which is consistent with the clinical features. Our clinical and experimental results consistently suggested that YWHAZ was a novel intellectual disability pathogenic gene.

IntroductionWith the advent of trio-based whole-exome sequencing, the identification of epilepsy candidate genes has become easier, resulting in a large number of potential genes that need to be validated in a whole-organism context. However, conducting animal experiments systematically and efficiently remains a challenge due to their laborious and time-consuming nature. This study aims to develop optimized strategies for validating epilepsy candidate genes using the Drosophila model.MethodsThis study incorporate behavior, morphology, and electrophysiology for genetic manipulation and phenotypic examination. We utilized the Gal4/UAS system in combination with RNAi techniques to generate loss-of-function models. We performed a range of behavioral tests, including two previously unreported seizure phenotypes, to evaluate the seizure behavior of mutant and wild-type flies. We used Gal4/UAS-mGFP flies to observe the morphological alterations in the brain under a confocal microscope. We also implemented patch-clamp recordings, including a novel electrophysiological method for studying synapse function and improved methods for recording action potential currents and spontaneous EPSCs on targeted neurons.ResultsWe applied different techniques or methods mentioned above to investigate four epilepsy-associated genes, namely Tango14, Klp3A, Cac, and Sbf, based on their genotype-phenotype correlation. Our findings showcase the feasibility and efficiency of our screening system for confirming epilepsy candidate genes in the Drosophila model.DiscussionThis efficient screening system holds the potential to significantly accelerate and optimize the process of identifying epilepsy candidate genes, particularly in conjunction with trio-based whole-exome sequencing.

Epileptic encephalopathy is a common devastating epilepsy with etiologies remain largely elusive, despite application of whole gene/exon sequencing on large cohorts. This study targeted on childhood epileptic encephalopathy, typically Lennox-Gastaut syndrome that is featured by age-dependent onset and characteristic clinical manifestations. Trio-based whole-exome sequencing with individualized analysis before statistic studies was employed, including individualized analysis on each trio by explainable inheritance origin and stratified frequency filtration and on each gene from four aspects. This study identified three novel candidate genes in 235 patients, includingSBF1withde novovariants in three cases,CELSR2with biallelic recessive variants in eight cases, andTENM1with X-linked recessive variants in six cases. These genes presented significant repetitiveness, including significant higher excess ofde novovariants inSBF1and excess of biallelic variants inCELSR2, and aggregated frequencies of variants inSBF1,CELSR2, andTENM1. The frequency of compound heterozygous/homozygousCELSR2variants in the cases was significantly higher than that in the asymptomatic parent-controls. Further experiments with knock-down/knock-out of these genes showed increased seizure-like behavior and increased firing of excitatory neurons. This study highlights the implication of phenotype sub-classification, individualized analysis in combination with statistic studies, and use of controls for compound heterozygous variants.