First-tier versus last-tier trio whole-genome sequencing for the diagnosis of pediatric-onset rare diseases

Despite advances in diagnostics, children with rare genetic disorders still face extended diagnostic odysseys, delaying appropriate clinical management, and placing burdens on families and healthcare resources. Whole-genome sequencing (WGS) offers a more comprehensive interrogation of the genome than other genetic tests, but its use in clinical practice remains limited. This study compared diagnostic rates, turnaround times, and clinical utility of first-tier versus last-tier trio-WGS for patients with suspected genetic pediatric-onset conditions, including 97 critical and 104 non-critical patients. Eighty-five patients (42.3%), including 57 (58.8%) critical and 28 (26.9%) non-critical patients, received a molecular diagnosis. The diagnostic rate was higher for first-tier (57%) than for last-tier (32.8%) trio-WGS. Of 121 causative variants identified, 19.8% would have been missed by whole-exome sequencing. Laboratory processing time was 4 days for all patients. The clinical setting had the greatest impact on time to reporting, averaging 5 days for critical patients versus 74 days for outpatients. WGS results impacted clinical decision-making for 34% of all critical and 14.3% of WGS-positive non-critical patients. This is the first Italian clinical study to demonstrate the diagnostic and clinical utility of a genome-first approach for both critical and non-critical patients with suspected genetic pediatric-onset disorders and feasibility in a public healthcare system.