Resolving Diagnostic Uncertainty in Neurodevelopmental Disorders Using Exome Sequencing Supported by Literature-Based
Danijela Krgovic1,2, Peter Gradisnik3, Andreja Osterc Koprivsek3
1University Institute of Genetic Diagnostics, University Medical Centre Maribor, 2000 Maribor, Slovenia.
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Background: 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. Methods: 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. Results: 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 KMT5B, TANC2, SPTBN1, and CHD4, 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. Conclusions: 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.
