Work

Addressing Interpretative Pitfalls in Neurodevelopmental Disorder Diagnostics: The NeuroWES Macedonia Experience

Year: 2026

Type: article

Abstract: Neurodevelopmental disorders (NDDs) are among the most genetically complex human conditions, yet even in the era of routine exome sequencing (ES), a large fraction of patients remains without a molecular diagnosis. Here, we analyzed 203 unrelated families from NeuroWES‐Macedonia, the first national genomic initiative in North Macedonia, using a fully integrated diagnostic strategy that combined trio‐based ES, copy number variant (CNV) analysis, RNA studies, and DNA methylation (episignature) profiling. Rather than focusing solely on diagnostic yield, we systematically examined recurrent interpretative failure modes that lead to missed, delayed, or incomplete diagnoses in routine clinical genomics. Likely pathogenic or pathogenic variants were identified in 29.6% (60/203) of probands, with diagnostic yield rising to 62.3% (38/61) in syndromic NDDs. However, many of the most instructive cases lay beyond conventional Mendelian expectations. We uncovered cryptic splicing defects evade in silico prediction, low‐level parental and proband mosaicism, multilocus genomic disease, and inherited pathogenic variants concealed by subtle or unrecognized parental phenotypes. Epigenomic profiling proved particularly powerful in resolving cases involving chromosomal abnormalities, mosaic aneuploidies, and overlapping syndromes, allowing us to disentangle primary from secondary molecular drivers. Beyond improving diagnostic understanding, analysis of this cohort expanded the mutational and phenotypic spectra of known NDD genes and yielded novel candidate disease genes, several of which were independently validated through international data sharing. Importantly, multiple cases demonstrated how rigid inheritance assumptions, reliance on de novo filtering, and compartmentalized analysis of SNVs and CNVs can actively obscure true disease mechanisms—even when high‐quality sequencing data are available. Together, our findings show that the persistent diagnostic gap in NDDs is driven not only by missing genes but also by systematic blind spots in variant interpretation. Integrating multiomic data with phenotype‐driven, flexible analytic frameworks is essential to unlock the full potential of genomic medicine in NDDs.

Source: Human Mutation

DOI: 10.1155/humu/7201395