Trio Genome Sequencing Identifies Diagnostic and Candidate Genes in Neurodevelopmental Disorder Unresolved by Prior Testing

<jats:p>Approximately 75% of individuals with neurodevelopmental disorders (NDD) remain without a molecular diagnosis after first-tier genetic testing, and a substantial share of that gap reflects the pace of gene-disease discovery rather than sequencing technology alone (Stefanski et al., 2021). Here we report trio genome sequencing in 36 probands with NDD or epilepsy who remained molecularly unsolved despite prior genetic testing. Likely pathogenic variants were identified in thirteen probands (36%): eight confirmed diagnoses and five high-priority findings pending Sanger validation, including a deep intronic PPP2R5C variant that extends the gene's recognised mutational spectrum. A further 19 candidate genes without an established disease association were prioritised by evidence score, including PRKAR1B, identified independently as a de novo duplication in two unrelated probands but not counted as a diagnosis pending clarification of its disease mechanism. Grouping all 34 findings by functional theme showed genes involved in transcription and chromatin regulation as the largest category. Only 9% of findings were in genome regions inaccessible to exome sequencing, indicating that most diagnostic value reflected thorough, trio-based analysis rather than access to genome-specific sequence. These findings support genome sequencing as a second-tier diagnostic step after negative prior testing, while showing that reassessing gene-disease validity, not sequencing technology, accounted for part of this yield.</jats:p>