Abstract: TH-PO579
Functional Impact of COL4A3/4A4 Variants in Patients with Nephrotic Syndrome
Session Information
- Glomerular Diseases: Omics, Biomarkers, and Tools
October 24, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Lai Yee, Jennifer, University of Michigan, Ann Arbor, Michigan, United States
- Glaser, Veronica, University of Michigan, Ann Arbor, Michigan, United States
- Fermin, Damian, University of Michigan, Ann Arbor, Michigan, United States
- Rheault, Michelle N., University of Minnesota Medical School, Minneapolis, Minnesota, United States
- Fornoni, Alessia, University of Miami, Coral Gables, Florida, United States
- Kitzman, Jacob O., University of Michigan, Ann Arbor, Michigan, United States
Background
The increasing prevalence of genetic testing in patients with kidney disease has led to the frequent identification of variants in COL4A3/4A4, which are vital components of the kidney's glomerular basement membrane. However, the diverse clinical manifestations of these variants remain poorly understood, and the lack of specific functional assays hampers the assignment of variant pathogenicity. We aim to investigate the functionality of COL4A3/4 in a cell-based collagen trimerization assay and study its impact on disease in a longitudinal observational cohort with nephrotic syndrome (NEPTUNE).
Methods
Patients with a heterozygous COL4A3/4 variant (n=32 heterozygous, n=1 homozygous) have been identified in the NEPTUNE cohort. We employed an established split luciferase assay to test trimerization (intracellular signal) and secretory effect (extracellular signal) of these variants. We created several known pathogenic, benign, and rare COL4A3/4 variants found in NEPTUNE and cloned them into the corresponding expression constructs. Intracellular and extracellular NanoLuc luciferase activity was then measured in 293T cells. Finally, we compared these in vitro functional activities with the clinical outcomes of the patients by a descriptive analysis.
Results
Low luciferase activity, either intracellularly or extracellularly, corresponded well to the expression constructs bearing the known pathogenic variants. 3 patients’ COL4A3 variants were successfully cloned and suggested to have a trimerization defect (N=1), secretory defect (N=1), and mild abnormality (N=1 with APOL1 high-risk genotype) based on intracellular and extracellular luciferase activity. The patient with a variant of trimerization defect had a kidney biopsy showing Alport findings. Both patients with a variant of secretory defect and mild abnormality were pediatric patients with progressive kidney disease (eGFR < 60 at baseline or a 40% loss of eGFR during follow-up).
Conclusion
These findings underscore the potential of the split luciferase assay as an effective functional assay for distinguishing benign and pathogenic COL4A variants. This paves the way for future development of the split luciferase as a clinical test to inform the variant pathogenicity and prognosis, and can enable more precise disease classification to foster further research development in NEPTUNE.
Funding
- Other NIH Support