Clinical value of luciferase-based bioluminescence assay in diagnosis of Alport syndrome

TL;DR

This study shows that a bioluminescence assay can help diagnose Alport syndrome by evaluating uncertain genetic variants in children.

Contribution

The study introduces a functional split-luciferase assay to assess variants of uncertain significance in Alport syndrome genes.

Findings

Luminescence intensity of VUS plasmids was reduced by over 50% compared to wild-type plasmids.

The assay achieved 96.77% sensitivity and 100% specificity in distinguishing VUS from wild-type variants.

Abstract

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.

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.

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%.

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.

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