Functionally Resolving <i>WT1</i> Variants of Uncertain Significance in Nephrotic Syndrome
October 22, 2020 | 10:00 AM - 12:00 PM
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Functionally Resolving WT1 Variants of Uncertain Significance in Nephrotic Syndrome
- Podocyte Biology
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1204 Podocyte Biology
- Lai Yee, Jennifer, University of Michigan, Ann Arbor, Michigan, United States
- Jia, Xiaoyan, University of Michigan, Ann Arbor, Michigan, United States
- Vega-Warner, V., University of Michigan, Ann Arbor, Michigan, United States
- Sampson, Matt G., Boston Children's Hospital, Boston, Massachusetts, United States
- Kitzman, Jacob O., University of Michigan, Ann Arbor, Michigan, United States
Jennifer Lai Yee,
Matt G. Sampson,
Jacob O. Kitzman,
Patients with Mendelian forms of nephrotic syndrome (NS) are likely to progress to end stage kidney disease. Because of the increased availability and promise to guide clinical decision, genetic screening among affected patients is proliferating. However, accurate attribution of pathogenicity to rare variants found during genetic screening remains challenging. WT1 is the second most common gene causing Mendelian NS. Therefore, this project aims to develop a model system to test the transcriptional activity of WT1 variants, as a first step towards high-throughput functional analysis to comprehensively classify variants in this key NS gene.
Wild-type and several bona fide pathogenic WT1 variants were tested for transcriptional activity in a standard dual-luciferase assay. Several cell lines including HeLa, HEK293, and HK2, were co-transfected with variant or wild-type WT1 and an NPHS1 promoter luciferase vector. Furthermore, potential WT1 target genes specific to HEK293 cells were identified by analyzing differential gene expression in RNA-Seq data of WT1 over-expressing HEK293 cells, in order to identify additional WT1-responsive promoters for use as WT1 activity reporters.
Overexpression of wild-type WT1 in HeLa cells and HEK cells increased expression of luciferase under the NPHS1 promoter by ~2-fold relative to truncated WT1. The luciferase activity of bona fide pathogenic WT1 variants was also significantly lower than the wild-type WT1 and bona fide benign WT1 variants (P<0.05). Overexpression of wild-type WT1 in HEK293 resulted in upregulation (log2 fold change>0.4, adjusted p<0.05) of IGF1R, EGFR, TGFB2. These candidates are being developed as WT1-responsive reporters.
Previous reports suggested NPHS1 promoter reporters as a model system to investigate mutant WT1 function. However, the transcriptional effect of WT1 was subtle and may be cell specific. Future work to establish a robust WT1 reporter is ongoing using cloned IGF1R, EGFR, TGFB2 promoters.
- Other NIH Support