Kidney Week

Abstract: SA-PO0128

A Dual-Luciferase Assay Platform for Functional Characterization of Variants of Uncertain Significance in the HNF1B Gene

Session Information

• ADPKD and Cystic Kidney Disease - 3
  October 24, 2026 | Location: Exhibit Hall A, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1201 Genetic Diseases of the Kidneys: Cystic (Monogenic)

Authors

• Zhang, Yaochun, National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Yaochun Zhang, PhD

• Lim, Si Ting, National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Si Ting Lim

• Ng, Jun Li, National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Jun Li Ng

• Koh, Chee Teck, National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Chee Teck Koh, MD

• Than, Mya, National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Mya Than, MBBS, MPH

• Chin, Hui-Lin L., National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Hui-Lin L. Chin, MBBS

• Ng, Kar Hui, National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Kar Hui Ng, MBBS

Background

HNF1B-related nephropathy is a common cause of bilateral renal hypo-dysplasia. Interpretation of variants of uncertain significance (VUS) in the HNF1B gene remains challenging. This study aims to establish a dual-luciferase assay platform for the functional characterization of VUS in HNF1B gene.

Methods

The HNF4A binding motif was subcloned upstream of the firefly luciferase coding sequence. This HNF4A-driven luciferase reporter was co-transfected into HEK293 cells with plasmid encoding either wild-type or mutant HNF4A. A CMV-driven renilla luciferase reporter was included as an internal control for normalization. Representative pathogenic variants were subsequently overexpressed in proximal tubular cells (HK2) for transcriptomic analysis.

Results

A total of 105 missense variants in HNF4A were selected from the gnomAD database, including 3 variants classified as benign/likely benign and 11 classified as pathogenic/likely pathogenic in ClinVar. The remaining 91 variants were either classified as VUS or had no ClinVar entry. Firefly luciferase activity was normalized to renilla luciferase activity and compared with the wild-type control. Using the firefly/renilla ratio of wild-type HNF4A as the 100% reference, and applying a 20% change threshold, all benign variants fell within the benign range of 80-120%. In contrast, pathogenic/likely pathogenic variants exhibited either increased or decreased HNF4A-targeted transcriptional activity. Among the 91 uncharacterized variants, 37 significantly increased firefly expression, with p.Arg295Cys showing the strongest gain-of-function effect at 419%, while 21 significantly decreased expression, with p.Thr415Ile showing the greatest loss-of-function effect at 23% of wild-type activity. These 58 variants could therefore be reclassified as pathogenic/likely pathogenic, whereas the remaining 33 variants remained within the benign range. Transcriptomic analysis of HK2 cells transfected with p.Arg295Cys and p.Ser547Phe, representing gain-of-function and loss-of-function variants, respectively, further confirmed the bidirectional regulatory effects of HNF4A mutants.

Conclusion

A dual-luciferase assay platform was established to functionally characterise VUS in the HNF1B gene. Using this platform, we evaluated more than 100 variants and found that pathogenic HNF1B variants can either upregulate or downregulate targeted gene expression.

Funding

• Government Support – Non-U.S.