Plasma DNA End-Motif Profiling as a Fragmentomic Marker in Cancer, Pregnancy, and Transplantation

Abstract

There is much recent research interest in the molecular characteristics of cell-free DNA (cf DNA) in plasma. One such characteristic is the fragmentation patterns of cfDNA, including information regarding fragment sizes (1), nucleosome relationships (2, 3), and end points (4, 5). This area of research can be broadly named "fragmentomics" (6). cfDNA molecules are known to circulate as short fragments (1, 7) originating from different cell types, including various normal organ systems aBstRact Plasma DNA fragmentomics is an emerging area of research covering plasma DNA sizes, end points, and nucleosome footprints. In the present study, we found a significant increase in the diversity of plasma DNA end motifs in patients with hepatocellular carcinoma (HCC). Compared with patients without HCC, patients with HCC showed a preferential pattern of 4-mer end motifs. In particular, the abundance of plasma DNA motif CCCA was much lower in patients with HCC than in subjects without HCC. The aberrant end motifs were also observed in patients with other cancer types, including colorectal cancer, lung cancer, nasopharyngeal carcinoma, and head and neck squamous cell carcinoma. We further observed that the profile of plasma DNA end motifs originating from the same organ, such as the liver, placenta, and hematopoietic cells, generally clustered together. The profile of end motifs may therefore serve as a class of biomarkers for liquid biopsy in oncology, noninvasive prenatal testing, and transplantation monitoring. SIGNIFICANCE: Plasma DNA molecules originating from the liver, HCC and other cancers, placenta, and hematopoietic cells each harbor a set of characteristic plasma DNA end motifs. Such markers carry tissueof-origin information and represent a new class of biomarkers in the nascent field of fragmentomics. Research.

Detection and characterization of jagged ends of double-stranded DNA in plasma

“…Furthermore, the shorter jagged ends were accompanied with a higher GC content (Supplemental Fig. S4), which was consistent with the fact that plasma DNA ends were enriched with the "CC" motif (Chandrananda et al 2015;Serpas et al 2019;Jiang et al 2020). Figure 5B shows that the mean lengths of jagged ends varied according to different sizes of plasma DNA fragments.…”

“…Such preferred ends were also shown to be related to the tissue of origin of plasma DNA (Chan et al 2016;Jiang et al 2018). Furthermore, certain motifs were preferentially present at the ends of plasma DNA molecules (Serpas et al 2019;Jiang et al 2020). Such end motif analysis revealed that DNASE1L3 (deoxyribonuclease 1-like 3), an endonuclease, plays an important role in the fragmentation of plasma DNA (e.g., the forming of the "CCCA" end motif of plasma DNA molecules), as elucidated by a Dnase1l3 deletion mouse model (Serpas et al 2019).…”

“…As tumor-derived DNA molecules contained higher jaggedness, we further explored if it was feasible to use plasma DNA jaggedness for detecting patients with HCC. We analyzed a cohort in a previously published study (Jiang et al 2020), consisting of healthy control subjects (n = 8), patients infected with chronic hepatitis B virus (HBV, n = 17), and patients with hepatocellular carcinoma (HCC, n = 34), using bisulfite sequencing. This experimental protocol involved a step of end repair with the use of unmethylated cytosines, allowing for calculating JI-U for each sample.…”

“…Such end motif analysis revealed that DNASE1L3 (deoxyribonuclease 1-like 3), an endonuclease, plays an important role in the fragmentation of plasma DNA (e.g., the forming of the "CCCA" end motif of plasma DNA molecules), as elucidated by a Dnase1l3 deletion mouse model (Serpas et al 2019). The down-regulation of DNASE1L3 in hepatocellular carcinoma (HCC) tumor tissues coincided with the reduction of the CCCA end motif in plasma of patients with HCC (Jiang et al 2020), resembling the consequence of Dnase1l3-deficient mice. In addition, different nucleases such as DNASE1 and DNASElL3 may be responsible for different cutting patterns with regard to the ends of cell-free DNA (Han et al 2020).…”

Implementation of the plasma MYCN/NAGK ratio to detect MYCN amplification in patients with neuroblastoma

“…Unfortunately, serial assessment of MYCN amplification of tumor is not possible due to the lack of primary tumor tissue and heterogeneity of tumor in NB. Nowadays, liquid biopsy test is emerging as a promising and repeatable method to examine tumor load in clinic [24,26,[44][45][46]. Using blood cfDNA from patients with NB sheds light on predicting MYCN amplification repeatedly [34][35][36].…”