Integrated Clinical-Genomic Reanalysis Identifies Diagnostic Gaps and Candidate Genes in a Pediatric Rare Disease...

Children with suspected rare genetic disorders often remain undiagnosed after exome, gene panel, or chromosomal microarray testing. We sought to define recurrent missed-diagnosis mechanisms and evaluate an integrated clinical-genomic reanalysis framework.

We studied 75 participants with rare or striking pediatric-onset phenotypes who remained undiagnosed after prior genetic testing. Trio genome sequencing evaluated SNVs, indels and copy-number variants. RNA sequencing was performed in 63 participants to assess aberrant expression and splicing, with targeted follow-up assays used selectively for prioritized variants. Clinician-genome scientist review integrated longitudinal phenotyping, physician input, and inheritance. Unresolved cases underwent research-level candidate prioritization.

Clinically reportable findings were returned in 9, including two with multilocus findings: 5 explained the primary phenotype and 4, while not fully explanatory, were clinically relevant and informing surveillance, management, or family follow-up. Previously missed findings reflected technical limitations, including deep intronic splice-altering variants supported by transcriptome analysis and a genomic deletion, and interpretive limitations related to incomplete phenotype integration or narrowly scoped prior testing. Research-level analysis prioritized six candidate genes, including biallelic ACHE variants with reduced enzymatic activity.

Integrated clinical-genomic reanalysis framework identified diagnostic gaps, recovered care-informing findings, and prioritized candidate genes for rare disease discovery.