Diagnostic Utility of Next-Generation Sequencing for Unconjugated Hyperbilirubinemia in Children

Purpose: Unconjugated hyperbilirubinemia can cause severe neurologic complications. Next-generation sequencing (NGS) is widely used for molecular diagnosis in pediatric disorders. This study evaluated the clinical features, diagnostic utility of NGS, and prognosis of children with unconjugated hyperbilirubinemia. Methods: Medical records of patients aged <18 years with unconjugated hyperbilirubinemia persisting >3 months from January 2015 to November 2024 were retrospectively reviewed. Hyperbilirubinemia was defined as total bilirubin >1.5 mg/dL. DNA was extracted from blood, and panel-based NGS targeting 96 genes was performed. Results: Among 98 patients, NGS was performed in 30 (30.6%), identifying pathogenic variants in 12 (40.0%). The most frequent diagnosis was Gilbert syndrome (20%), followed by hereditary spherocytosis (16.7%); one patient (3.3%) had Rotor syndrome. Among patients with hereditary spherocytosis, only one showed elevated reticulocyte counts, and none had anemia. Resolution of hyperbilirubinemia occurred in 34 patients (34.7%) with a median duration of 6.6 months. Persistent hyperbilirubinemia >1 year occurred in 35 patients (35.7%), although levels >5 mg/dL were not observed during the maximum follow-up period. Multivariate analysis showed peak bilirubin level (odds ratio, 1.65; 95% confidence interval, 0.087-2.895) was significantly associated with abnormal NGS results. Conclusion: NGS provides effective diagnosis for unconjugated hyperbilirubinemia, with a 40% yield. Hemolytic disease should be considered as an underlying cause, even without anemia.