ORIGINAL RESEARCH article
Front. Pediatr.
Sec. Pediatric Nephrology
Clinical value of luciferase‑based bioluminescence assay in diagnosis of Alport syndrome
Provisionally accepted
- Shanghai Children's Hospital, Shanghai, China
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Objectives: Alport syndrome (AS) is an inherited kidney disorder caused by pathogenic variants in COL4A3, COL4A4, or COL4A5. In this study, we aim to apply a split‑luciferase bioluminescence assay to functionally assess COL4A3, COL4A4, or COL4A5 variants of uncertain significance (VUS) identified in pediatric patients with Alport syndrome, and to explore its value in supporting variant interpretation and diagnostic evaluation. Methods: A retrospective analysis included 31 children who met established clinical and pathological diagnostic criteria for Alport syndrome, but in whom genetic testing identified VUS in COL4A3, COL4A4, or COL4A5. Genomic DNA was analyzed using next-generation sequencing (NGS) to identify variant loci. Recombinant plasmids corresponding to the identified variants were constructed and transfected into HEK293T cells. The split‑luciferase bioluminescence assay (NanoBiT® system) was employed to measure luminescence signals in living cells. Luminescence intensity was compared between the VUS-associated plasmids and the wild-type (WT) plasmid, and the sensitivity and specificity of this technique were evaluated. Results: The luminescence intensity of all 31 plasmids carrying VUS identified in children with Alport syndrome was reduced by more than 50% compared with the WT plasmid group (P < 0.01, n = 3). When the luminescence intensity was below 397.8, the sensitivity for distinguishing VUS from WT was 96.77%, and the specificity was 100%. Conclusions: The split-luciferase bioluminescence assay was successfully applied as an in vitro functional approach to assess COL4A3, COL4A4, or COL4A5 VUS, and may reliably assist in the diagnostic evaluation of pediatric patients with suspected Alport syndrome whose genetic testing reveals VUS, particularly in cases where pathological assessment is unavailable or not feasible.
Keywords: Alport syndrome, diagnosis, Genetic Variation, Luciferases, Pediatrics
Received: 16 Sep 2025; Accepted: 27 Jan 2026.
Copyright: © 2026 Cai, Lin, Kuang, Sun, Li, Hu and Huang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Wenyan Huang
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