Identifying genetic causes and establishing a diagnostic approach for WES-negative pediatric population with...

Global developmental delay (GDD) and intellectual disability (ID) are frequently caused by genetic factors, yet many patients remain undiagnosed even after whole exome sequencing (WES). This study aimed to apply Optical Genome Mapping (OGM) and Illumina Complete Long Reads (ICLR) in pediatric patients with unexplained GDD/ID after WES and propose a practical diagnostic strategy for clinical implementation. We conducted OGM and ICLR on 87 pediatric patients with unexplained GDD/ID despite prior WES. Discordant cases underwent further validation using gap-PCR or PacBio long-read sequencing. A minigene assay was also performed to confirm the pathogenicity of an intronic variant. Of the 87 patients, 6 were found to carry pathogenic or likely pathogenic variants, including 4 structural variants (SVs) and 2 single nucleotide variants (SNVs). OGM and ICLR provided additional diagnostic yields of 4.71% and 6.98%. OGM was effective in detecting complex rearrangements, whereas ICLR performed well in cases with overlapping structural variants. For all SV burden, ICLR detected 8 SVs (mean 0.09 ± 0.33 per sample), and OGM identified 8 SVs (mean 0.09 ± 0.29 per sample), showing comparable results. This study demonstrates the complementary utility of ICLR and OGM in detecting diverse classes of pathogenic variants in GDD/ID. ICLR was advantageous for detecting non-coding SNVs, as well as for providing accurate breakpoint resolution in SVs, while OGM was effective for complex rearrangements and repetitive regions. These findings support a stepwise diagnostic strategy in which ICLR may be considered as an early second-tier test for WES-negative GDD/ID cases.