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Fragmentomics of urinary cell-free DNA in nuclease knockout mouse models
Meihui Chen1,2,3, Rebecca W Y Chan1,2,3, Peter P H Cheung2,3
1Centre for Novostics, Hong Kong Science Park, Pak Shek Kok, New Territories, Hong Kong SAR, China.
Abstract:
Urinary cell-free DNA (ucfDNA) is a potential biomarker for bladder cancer detection. However, the biological characteristics of ucfDNA are not well understood. We explored the roles of deoxyribonuclease 1 (DNASE1) and deoxyribonuclease 1-like 3 (DNASE1L3) in the fragmentation of ucfDNA using mouse models. The deletion of Dnase1 in mice (Dnase1-/-) caused aberrations in ucfDNA fragmentation, including a 24-fold increase in DNA concentration, and a 3-fold enrichment of long DNA molecules, with a relative decrease of fragments with thymine ends and reduction of jaggedness (i.e., the presence of single-stranded protruding ends). In contrast, such changes were not observed in mice with Dnase1l3 deletion (Dnase1l3-/-). These results suggested that DNASE1 was an important nuclease contributing to the ucfDNA fragmentation. Western blot analysis revealed that the concentration of DNASE1 protein was higher in urine than DNASE1L3. The native-polyacrylamide gel electrophoresis zymogram showed that DNASE1 activity in urine was higher than that in plasma. Furthermore, the proportion of ucfDNA fragment ends within DNase I hypersensitive sites (DHSs) was significantly increased in Dnase1-deficient mice. In humans, patients with bladder cancer had lower proportions of ucfDNA fragment ends within the DHSs when compared with participants without bladder cancer. The area under the curve (AUC) for differentiating patients with and without bladder cancer was 0.83, suggesting the analysis of ucfDNA fragmentation in the DHSs may have potential for bladder cancer detection. This work revealed the intrinsic links between the nucleases in urine and ucfDNA fragmentomics.
Insights
Urinary cell-free DNA (ucfDNA) fragmentation is influenced by deoxyribonuclease 1 (DNASE1), not DNASE1L3. Altered ucfDNA fragmentation patterns, particularly within DNase I hypersensitive sites (DHSs), show promise for bladder cancer detection.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Urinary cell-free DNA (ucfDNA) shows potential as a non-invasive biomarker for bladder cancer.
- The precise mechanisms governing ucfDNA fragmentation remain incompletely understood.
Purpose of the Study:
- To investigate the roles of deoxyribonuclease 1 (DNASE1) and deoxyribonuclease 1-like 3 (DNASE1L3) in ucfDNA fragmentation.
- To explore the potential of ucfDNA fragmentation patterns within DNase I hypersensitive sites (DHSs) for bladder cancer detection.
Main Methods:
- Utilized mouse models with targeted deletions of Dnase1 and Dnase1l3 genes.
- Analyzed ucfDNA fragmentation characteristics, including DNA concentration, fragment length, and end-specific features.
- Compared DNASE1 and DNASE1L3 protein levels and enzymatic activity in urine and plasma.
- Examined the distribution of ucfDNA fragment ends within DHSs in both mouse models and human bladder cancer patients.
Main Results:
- Deletion of Dnase1, but not Dnase1l3, significantly altered ucfDNA fragmentation in mice, increasing DNA concentration and favoring longer molecules.
- DNASE1 exhibited higher concentration and activity in urine compared to DNASE1L3.
- A significant increase in ucfDNA fragment ends within DHSs was observed in Dnase1-deficient mice.
- Patients with bladder cancer showed a lower proportion of ucfDNA fragment ends within DHSs compared to healthy individuals, with an AUC of 0.83 for differentiation.
Conclusions:
- DNASE1 is a key nuclease responsible for ucfDNA fragmentation in urine.
- The fragmentation patterns of ucfDNA, specifically the proportion of fragment ends within DHSs, represent a promising biomarker for non-invasive bladder cancer detection.
