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Abstract: TH-PO781

Insertions and Deletions (InDels) in the Non-Translated Human Genome Upstream of ZHX2 Alter ZHX2 mRNA Expression in MCD and FSGS

Session Information

Category: Glomerular Diseases

  • 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix

Authors

  • Chugh, Sumant S., Rush University Medical Center, Chicago, Illinois, United States
  • Das, Ranjan, Rush University Medical Center, Chicago, Illinois, United States
  • Del Nogal Avila, Maria, Rush University Medical Center, Chicago, Illinois, United States
  • Kharlyngdoh, Joubert Banjop, Rush University Medical Center, Chicago, Illinois, United States
  • Donoro blazquez, Hector, Rush University Medical Center, Chicago, Illinois, United States
  • Molina-Jijon, Eduardo, Rush University Medical Center, Chicago, Illinois, United States
  • Clement, Lionel C., Rush University Medical Center, Chicago, Illinois, United States
  • Mace, Camille E., Rush University Medical Center, Chicago, Illinois, United States
  • Avila-Casado, Carmen, University Health Network, University of Toronto, Toronto, Ontario, Canada

Group or Team Name

  • Glomerular Disease Therapeutics Laboratory
Background

ZHX proteins, especially ZHX2, play a critical role as transcriptional regulators of human and experimental podocyte disease. Multiple groups conducting whole exome sequencing were unable to find significant mutations in these genes in human glomerular disease. We sequenced the genome upstream of ZHX2 and the intronic sequence looking for insertions and deletions that may induce DNA conformational changes, resulting in altered ZHX2 expression.

Methods

The 1.3 million bp region between the beginning of the immediate upstream gene HAS2 and the end of ZHX2 was sequenced in 28 patients with nephrotic syndrome (8 MCD, 2 FSGS tip lesion, 8 FSGS with mutations in slit diaphragm genes, 4 recurrent FSGS,2 recurrent non-HIV collapsing glomerulopathy, and 4 Hodgkin disease with nephrotic syndrome) and 27 healthy controls using Agilent Custom capture and high throughput Illumina sequencing to obtain about 8 million sequences per sample. The Qiagen Biomedical Genomics Workbench software was used to identify InDels > 3 bp and > 20 sequences present exclusively in the patient population. One of the InDels identified was replicated in cultured podocytes using CRISPR Cas9 technology to study changes in ZHX2 expression.

Results

We identified 5 InDels (size range 6 to 67) shared by than one patient and 40 others (size range 4 to 133) present in a single patient. These InDels were absent in controls and the 1000 genomes project. Patients with MCD and FSGS tip lesion had a high percentage of deletions (approximately 80%), whereas those with other forms of FSGS had mostly insertions (approximately 66%). Significance of these indels was verified by inserting one of these indels upstream of ZHX2 in single cell derived immortalized human podocytes by CRISPR-Cas9 approach. Podocytes carrying this InDel developed reduced ZHX2 expression.

Conclusion

Insertions and deletions upstream of the ZHX2 gene are commonly present in patients with MCD and FSGS patients and alter ZHX2 mRNA expression.

Funding

  • NIDDK Support