Dynamics of Specific cfDNA Fragments in the Plasma of Full Marathon Participants.
Author(s): Sugasawa T, Fujita SI, Kuji T, Ishibashi N, Tamai K, Kawakami Y, Takekoshi K
Publication: Genes (Basel), 2021, Vol. 12, Page
PubMed ID: 33946330 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare cell-free DNA (cfDNA) concentration and fragment size in plasma obtained pre-marathon and 0 h, 2 h, and 1 day after completion of the marathon. Additionally, the authors compared changes in levels of the most highly expressed specific cfDNA (spcfDNA-1) over the time course with changes observed in white blood cell (WBC) counts, myoglobulin levels, and creatine kinase activity (CK).
Conclusion of Paper
The cfDNA concentration was approximately 10-fold higher immediately post-marathon than pre-marathon but was comparable to baseline by 2 h post-marathon and was 38% of baseline by 1 day post-marathon. The increase in cfDNA immediately post-marathon corresponded to a large increase in 150 bp fragments while the 2 h post-marathon specimens had much more DNA of 2-7 kb than the other specimens. The cfDNA with the highest number of reads compared to GAPDH was identified and termed scpfDNA-1. Levels of spcfDNA-1 were 2-fold and 3-fold higher at the 0 and 2 h post-marathon timepoints relative to pre-marathon but only 1.3-fold higher than pre-marathon at the 1 day timepoint. WBC counts and myoglobin levels were strongly correlated with those of spcfDNA, with much higher levels at the 0 and 2 h post-marathon timepoints and levels only slightly higher than pre-marathon found at the 1 day timepoint. In contrast, CK levels increased progressively over the course of the timepoints but only weakly correlated with CK (R=0.275, P<0.01). CK increased progressively, with the highest levels found 1 day post-marathon and only a weak correlation to spcfDNA-1 levels were observed.
Studies
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Study Purpose
The purpose of this study was to compare cfDNA concentration and fragment size in plasma obtained pre-marathon and 0 h, 2 h, and 1 day after completion of the marathon. Additionally, the authors compared changes in levels of spcfDNA-1 over the time course with changes observed in WBC counts, myoglobulin levels, and CK. EDTA blood was obtained from 26 healthy males before marathon running (baseline), immediately after finishing marathon, and 2 h and 1 day after running the marathon. Only water was consumed in the 2 h after running the marathon. Blood was centrifuged at 3000 rpm for 15 min at 4°C and the resultant plasma stored at -80°C until analysis. White blood cell counts, plasma myoglobin, and creatinine kinase activity were measured by a clinical laboratory. Plasma pools were generated by mixing an equal volume of plasma from each participant for a given timepoint. DNA was extracted from plasma pools and individual specimens using NucleoSnap cfDNA Kit. cfDNA concentration and fragment size from the pooled plasma were analyzed using the Agilent High Sensitivity DNA Kit with a bioanalyzer. Extracted DNA was stored at -20°C. An NGS library was prepared from extracted pooled pre-marathon plasma DNA using SMARTer ThruPLEX Plasma-seq Kit and pair-end sequenced using a NextSeq 500 System. Specific cfDNAs were identified using the Transcription Factor ChIP-Seq program on CLC GenomicsWorkbench 20.0.3. spcfDNA and GAPDH were further quantified by real-time qPCR.
Summary of Findings:
The cfDNA concentration was approximately 10-fold higher immediately post-marathon than pre-marathon but was comparable to baseline by 2 h post-marathon and was 38% of baseline by 1 day post-marathon. The increase in cfDNA immediately post-marathon corresponded to a large increase in 150 bp fragments while the 2 h post-marathon specimens had much more DNA of 2-7 kb than the other specimens. The top five significant sequences identified by next-generation sequencing ranged from 467.8 to 17,606-fold higher than GAPDH. The median CT value was found to be much lower for the top sequence (spcfDNA-1) than GAPDH (20.4 versus 34.4). This corresponded to a median of 16,638-fold more copies of scpfDNA-1 than GAPDH (P<0.001). The standard deviation of the CT was also lower for spcfDNA-1 than GAPDH (0.398 versus 0.062, P<0.001). Levels of spcfDNA-1 were 2-fold and 3-fold higher at the 0 and 2 h post-marathon timepoints relative to pre-marathon (P<0.001, both) but only 1.3-fold higher than pre-marathon at the 1 day timepoint (P<0.05). WBC counts and myoglobin levels mirrored those for spcfDNA, with much higher levels at the 0 and 2 h post-marathon timepoints (P<0.001, all) and levels only slightly higher than pre-marathon found at the 1 day timepoint (P,0.05 and P<0.01, respectively). spcfDNA levels were strongly correlated with WBC counts and myoglobin levels (R=0.832, P<0.0001 and R-0.753, P<0.0001, respectively) but only weakly correlated with CK (R=0.275, P<0.01). CK increased progressively over the course of the timepoints with the highest levels found 1 day post-marathon (P<0.001).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Time of biospecimen collection Before marathon
Immediately after marathon
2 h post-marathon
1 day post-marathon
Real-time qPCR Specific Targeted nucleic acid spcfDNA-1
GAPDH
Real-time qPCR Specific Technology platform NGS
Clinical chemistry markers