Accurate and cost-effective workflow integrating trio pooled-WES for novel gene discovery in neurodevelopmental...

The broad genetic heterogeneity of neurodevelopmental disorders (NDDs) makes their molecular diagnosis particularly challenging. In this context, Whole-Exome Sequencing (WES), specifically in a trio-based design, is a powerful strategy due to its ability to detect de novo variants, which are a major contributor to NDDs. However, its clinical implementation is often limited by its associated cost. In this study, we applied a sequential diagnostic workflow to a cohort of 221 individuals with syndromic NDDs and prior negative results from targeted sequencing. The workflow integrates initial solo-WES, followed by a second-tier trio-WES using pooled parental DNA (trio pooled-WES). Overall, this workflow achieved a diagnostic yield of 20.98% and led to the identification of 13 novel candidate genes. The pooling strategy was optimized and validated, demonstrating that trio pooled-WES retains the main advantages of conventional trio-WES while substantially reducing sequencing costs. These results support its implementation as a clinically applicable approach for the genetic diagnosis of NDDs.