Abstract: SA-PO1173
Missense Mutations in PKD1 Lead to Reduced Surface Localization of the Polycystins in ADPKD
Session Information
- NIDDK KUH Summer Undergraduate Research Program Posters
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1001 Genetic Diseases of the Kidneys: Cystic
Author
Group or Team Name
- Sanjna Girdhar. Mayo Clinic, Rochester, MN.
Background
Autosomal Dominant Polycystic Kidney Disease (ADPKD), characterized by the development of fluid filled cysts, is the fourth leading cause of renal failure in the US and is mainly caused by mutations in the genes PKD1 or PKD2 encoding the proteins Polycystin1 (PC1) and Polycystin2
(PC2) respectively. Localization of PC1 and PC2 to the cell surface is important for their function in cells, with a positive correlation between reduced surface protein levels to disease severity. The objective of this project was to quantify surface localization of a number of missense variant PC1 proteins, and combination of variants.
Methods
The single PC1 variants (p.Arg3277Cys, p.Met3346Leu, and p.Thr3270Met) were created by site directed mutagenesis and introduced into the full length PC1 tagged construct. In addition, variant combinations were similarly generated (p.Thr3270Met/p.Arg3277Cys, p.Met3346Leu/p.Thr3270Met, and p.Arg3277Cys/p.Met3346Leu/p.Thr3270Met), mimicking a variant combination detected in three ADPKD families. Epithelial cells from the kidney were transfected with constructs containing these single and combination variants. Immunofluorescence staining of the transfected cells was used to visualize the surface localized proteins.
Results
The intensity of the immunofluorescence signal, as determined by flow cytometry, indicated the amount of protein properly localizing to the cell membrane. The single variants were found to have reduced surface localization compared to wild type, with the surface localization further lowered as the number of significant variants increased.
Conclusion
Therefore an in cis combination of missense variants can significantly reduce PC1 surface localization, showing that a combination of hypomorphic alleles can result in a fully inactivating allele.