Takenouchi-Kosaki syndrome (TKS), caused by the CDC42 c.191A>G (p.Tyr64Cys; Y64C) variant, characterized by a range of clinical manifestations, including developmental delay with intellectual disability (ID). While CDC42 is essential for neurogenesis, the mechanisms by which the Y64C mutation contributes to the neurological impairments observed in TKS patients remain unclear. This study aimed to investigate the functional impact of ectopically expressing the Y64C variant on the neurite outgrowth of neuroblastoma cells, Neuro2A. Neuro2A cells were transfected with the Y64C variant to assess changes in neurite outgrowth and cellular morphology. The cells were also treated with a lipidation inhibitor, GGTI-298, or CDC42 activity inhibitor, ML141 to evaluate its ability to ameliorate the mutation-induced phenotypes. Whole transcriptome sequencing and differentially expressed gene (DEG) analysis were performed between WT- and Y64C-expressing cells. The overexpression of the Y64C variant impaired neurite outgrowth and changed the cell morphology of Neuro2A cells. Both the neurite outgrowth defect and the enhanced membrane localization induced by Y64C overexpression were restored by GGTI-298 treatment but not by ML141. To explore the molecular basis of these phenotypes, we performed DEG analysis and identified 32 upregulated and 19 downregulated genes, including Smarca1. Consistent with the transcriptomic findings, Smarca1 protein expression was decreased in Y64C-expressing cells in comparison to WT. Treatment with GGTI-298 effectively preserved Smarca1 levels, whereas ML141 reduced its expression in both WT- and Y64C-expressing cells. The Y64C variant disrupts neurite outgrowth, attributable to altered CDC42 activity and localization. The restoration of neurite outgrowth by GGTI-298 highlights the importance of CDC42 signaling for neurite outgrowth in Neuro2A cells. Our findings raise the possibility that molecular disruptions caused by the Y64C mutation may contribute to the brain malformations and neurodevelopmental features observed in TKS.

Over the past few decades, advances in genomic analysis techniques and bioinformatics have enabled the identification of many new human monogenic diseases. In 2015, the Japan Agency for Medical Research and Development launched a national project for undiagnosed diseases called the Initiative on Rare and Undiagnosed Diseases (IRUD). Through this project, we identified Takenouchi-Kosaki syndrome (OMIM#616737), which is caused by specific pathogenic variants in CDC42, a critical regulator of diverse cellular functions, and is clinically characterized by intellectual disability and macrothrombocytopenia. In addition to the identification of disease-causing genes and new human monogenic disorders, significant progress has also been made in the clinical implementation of genomic medicine. In 2019, we launched a national project called Precise and Rapid Genetic Diagnosis and Treatability for Infants (Priority-i) to provide rapid genetic diagnosis for sick newborns in neonatal intensive care units. It is our mission to apply the benefits of the latest advances in genomic medicine to the clinical care of newborns and children.

F-Actin cytoskeleton remodeling is vital for cell migration, organ development and immune responses. The small GTPase CDC42, a key regulator of F-actin dynamics, cycles between inactive GDP- and active GTP-bound states. However, mechanisms governing CDC42 turnover and their biological significance remain unclear. Here we show that KLHL23-mediated polyubiquitylation of CDC42•GTP and RhoGDI-mediated sequestration of CDC42•GDP spatiotemporally co-inactivate CDC42, preserving membrane dynamics and homeostasis during migration. KLHL23-Cul3 acts as the E3 ligase for CDC42 degradation, with KLHL23 and RhoGDI competing for CDC42's switch II region, enhancing selectivity toward CDC42•GTP and CDC42•GDP, respectively. KLHL23 depletion disrupts membrane homeostasis, inducing excessive protrusions and promoting metastasis. Notably, the CDC42-Y64C germline variant in Takenouchi-Kosaki Syndrome escapes KLHL23-mediated degradation. Fluorescence resonance energy transfer assays reveal that KLHL23 and RhoGDI coordinately inactivate CDC42 in a spatiotemporal manner. These findings highlight the biological and clinical relevance of the KLHL23/RhoGDI-CDC42 axis, presenting new avenues for therapeutic exploration.

We present a 2-year-old male with bilateral iris and chorioretinal colobomas, speech delays, and facial and digital anomalies. Trio exome sequencing demonstrated a de novo, novel heterozygous variant, c.379G>A p.Glu127Lys in CDC42, conferring a diagnosis of Takenouchi-Kosaki syndrome. The p.Glu127Lys variant was not located in the same region as previously designated mutation classes for CDC42, and the patient's missense substitution was predicted to disrupt CDC42 interactions with Collybistin II and IQGAP1. As conditional knock-out mouse models have demonstrated coloboma in association with loss of Cdc42 expression, we conclude that the colobomas can be attributed to the CDC42 variant and that similar ocular anomalies are likely to be described with other Rho GTPases in the future.