Abstract: SA-PO1195
Novel Single-Cell Sequencing Methods for Detection of Mosaic Chromosomal Alterations in the Kidneys
Session Information
- CKD: Mechanisms - 3
October 26, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2303 CKD (Non-Dialysis): Mechanisms
Authors
- Wang, Gary, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States
- Chen, Xinyi Emilia, University of Pennsylvania Wharton School, Philadelphia, Pennsylvania, United States
- Verma, Amit, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States
- Zhang, Nancy, University of Pennsylvania Wharton School, Philadelphia, Pennsylvania, United States
- Wilson, Parker C., University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States
Background
Mosaic chromosomal alterations (mCA) are a type of somatic mosaicism characterized by large chromosomal gains or losses. We previously reported that mosaic loss of Y chromosome (LOY) increases with age and is enriched in an injured population of proximal tubule cells. However, the majority of these cells do not have LOY, which raises the possibility there are additional mCA we cannot detect. Here, we jointly profile genomic DNA and RNA by single-cell sequencing to unpack inaccessible chromatin regions and improve sensitivity for detection of mCA in the kidney.
Methods
We performed DEFND-seq (Olsen et al., bioRxiv 2023) and single cell multiome ATAC + gene expression (10X Genomics) on nuclei isolated from male rat kidney cortex aged 16 to 82 weeks. Split samples were subject to either standard nuclei processing (10X Multiome) or pretreatment with lithium diiodosalicylate to deplete nucleosomes. We generated 8 droplet-based single-cell libraries (DEFND = 4; Multiome = 4) and called mCA using a negative binomial model adjusting for cell-specific factors. We validated sex chromosome mCA using a 3-color digital PCR assay.
Results
A total of 40,031 cells passed filters and the number was similar between DEFND and Multiome libraries. DEFND had a 25-50% increase in genome-wide coverage compared to Multiomes, indicating we were able to “open” inaccessible regions for mCA analysis. We estimated mCA abundance for cell types and observed an increase in chromosome X (chrX) mCA compared to other chromosomes. The proportion of cells with a chrX mCA was higher in DEFND libraries, suggesting increased sensitivity in the DEFND assay. In contrast, chromosome 1 had a low probability of mCA and the proportion of cells was unchanged (Figure 1A). Approximately 8% of proximal tubule had a chrX mCA and enrichment analysis shows upregulation of oxidative phosphorylation and epithelial migration pathways (Figure 1B).
Conclusion
We hypothesize that sex chromosome mCA are enriched in the proximal tubule and are associated with increased oxidative stress. Our approach can be used to study the role of mCA as biomarkers of aging and kidney disease progression.
Funding
- NIDDK Support