Resolving Diagnostic Uncertainty in Neurodevelopmental Disorders Using Exome Sequencing Supported by Literature-Based Multi-Omics Evidence

<i>Background</i>: Neurodevelopmental disorders (NDDs) are genetically heterogeneous, and exome sequencing (ES) is now a first-line diagnostic tool. However, many patients receive variants of uncertain significance (VUSs) or inherited variants with incomplete penetrance, limiting clinical interpretation. Emerging multi-omics evidence from the literature can support the interpretation of novel and rare variants, helping to refine classification in selected cases. <i>Methods</i>: We assessed 20 patients and their parents referred for genetic testing for NDDs. ES was performed, followed by ACMG/ACGS-based variant classification, segregation analysis, and targeted literature review. Variants were included when deemed plausible contributors to the phenotype by a multidisciplinary team. Gene-level constraint metrics, in silico predictions, and emerging multi-omics evidence from the literature were integrated to support interpretation. <i>Results</i>: Across 18 NDD-associated genes, we identified 20 rare variants: Three pathogenic (P), nine likely pathogenic (LP), and eight VUSs. All P and most LP variants were de novo. Inherited variants, particularly in <i>KMT5B</i>, <i>TANC2</i>, <i>SPTBN1</i>, and <i>CHD4</i>, highlighted challenges related to incomplete penetrance. Two patients had dual molecular diagnoses. Several VUSs were supported by literature-derived transcriptomic, proteomic, or model-system evidence. <i>Conclusions</i>: This cohort underscores ongoing challenges in interpreting VUSs and inherited variants in NDDs. Integrating genomic findings with published multi-omics data enhances variant interpretation, reveals mechanistic insights, and strengthens diagnostic confidence, supporting broader adoption of multi-omics approaches in rare NDD evaluation.