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Abstract: SA-PO743

A Missense Mutation in Zinc Finger 4 of WT1 Might Lead to Focal Segmental Glomerular Sclerosis due to Its Mislocalization and Downstream Dysregulation

Session Information

Category: Glomerular Diseases

  • 1304 Glomerular Diseases: Podocyte Biology

Authors

  • Kanamori, Toru, Tokyo Ika Shika Daigaku Daigakin Ishigaku Sogo Kenkyuka, Tokyo, Japan
  • Udagawa, Tomohiro, Tokyo Ika Shika Daigaku Daigakin Ishigaku Sogo Kenkyuka, Tokyo, Japan
  • Murakoshi, Miki, Tokyo Ika Shika Daigaku Daigakin Ishigaku Sogo Kenkyuka, Tokyo, Japan
  • Adachi, Eriko, Tokyo Ika Shika Daigaku Daigakin Ishigaku Sogo Kenkyuka, Tokyo, Japan
  • Okutsu, Mika, Tokyo Ika Shika Daigaku Daigakin Ishigaku Sogo Kenkyuka, Tokyo, Japan
  • Mori, Takayasu, Tokyo Ika Shika Daigaku Daigakin Ishigaku Sogo Kenkyuka, Tokyo, Japan
  • Sohara, Eisei, Tokyo Ika Shika Daigaku Daigakin Ishigaku Sogo Kenkyuka, Tokyo, Japan
  • Uchida, Shinichi, Tokyo Ika Shika Daigaku Daigakin Ishigaku Sogo Kenkyuka, Tokyo, Japan
Background

Wilms’ tumor suppressor gene 1 (WT1) is associated with the development of the urogenital system and regulates genes involved in sex differentiation and determination. WT1 encodes four zinc finger domains (ZF). Inactivation in ZF2 or 3 of WT1 causes Denys-Drash syndrome (46, XY disorder of sex development [DSD] and renal disorders) and in intron 9 of WT1 causes Frasier syndrome (genital tract anomalies and focal segmental glomerulosclerosis [FSGS]). Eozenou et al. reported that mutations in ZF4 of WT1 cause 46, XX DSD (ProNAS. 2020, 13680). There is insufficient evidence associating ZF4 variants and nephropathy.

Methods

A 15-year-old boy presented with proteinuria, renal dysfunction, and undescended testis. He was diagnosed with FSGS by renal biopsy at 16 years and started hemodialysis at 20 years of age. To analyze the pathogenic mechanism of the mutations (WT1 R495Q) found in this FSGS patient, we evaluated localization of WT1 and other podocyte slit-diaphragm proteins by immunofluorescence staining using C-terminal antibodies of a kidney section. HEK293T cells were transfected with expression constructs to encode the wild type or R495Q WT1 protein. To examine the effect of WT1 R495Q on the regulation of key podocyte genes, we quantitatively assessed messenger ribonucleic acid (mRNA) expression levels isolated from wild type or mutation-expressing HEK293 cells by qPCR.

Results

Immunofluorescence staining showed that in contrast to the cytoplasmic localization of WT1 in a boy with non-genetic nephrotic syndrome, WT1 was localized to podocyte cytoplasm and nucleus. We found less expression of the NPHS1 gene in the case of transfection with WT1 R495Q compared to wild types. NPHS1 mRNA expressed approximately 25-fold whereas NPHS mRNA expressed approximately 10-fold compared to the control. When wild type and mutant WT1 were transfected with the same dose, the expression of NPHS1 was the same as when only the mutant was transfected.

Conclusion

We demonstrate that WT1 R495Q mutation affects WT1 protein localization and dysregulates NPHS1 expression in podocytes. Our data suggests the loss of function mutation had a dominant negative effect in podocytes.

Funding

  • Government Support – Non-U.S.