Exploring EPHB4 in Gait Disorders: VUS Detection and Phenotypic Characterization in a Toe-Walking Cohort

Background: Idiopathic toe walking (ITW) is a gait anomaly with suspected genetic origins, though underlying mechanisms remain poorly understood. Although the established role of ephrin type-B receptor 4 (EPHB4) involves mainly vascular development, in this study we focused whether the detected variants might correlate with clinical findings in idiopathic toe walkers cohort where EPHB4 variants were identified. Methods: We retrospectively analyzed clinical and genetic data from 20 toe-walking patients harboring EPHB4 variants detected using 49-gene Next-Generation Sequencing (NGS) panel. Results: Genetic analysis identified 18 distinct EPHB4 variants, all classified as variants of uncertain significance (VUS) per American College of Medical Genetics and Genomics (ACMG) guidelines Common clinical features included: • Pes cavus (40%) • Clinodactyly/brachydactyly (40%) • Speech/language delays (40%) Notably, only 10% showed venous anomalies typically associated with EPHB4 mutations . Conclusions: While this study reports an unexpected frequency of EPHB4 variants in ITW patients, these findings should be interpreted cautiously given: - The VUS classification of all variants - Lack of clear genotype-phenotype correlation - Potential ascertainment bias - Further investigation through functional studies and familial segregation analysis is needed to determine whether these observations represent a true biological signal or incidental findings. This work highlights the complexity of studying genetic factors in common pediatric conditions like ITW. Clinial trial registration: not applicable.Retrospective cohort analysis of existing clinical data. Not prospectively registered as no interventions were performed.