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1.
Proc Natl Acad Sci U S A ; 120(17): e2220982120, 2023 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-37075072

RESUMO

Cell-free DNA (cfDNA) fragmentation is nonrandom, at least partially mediated by various DNA nucleases, forming characteristic cfDNA end motifs. However, there is a paucity of tools for deciphering the relative contributions of cfDNA cleavage patterns related to underlying fragmentation factors. In this study, through non-negative matrix factorization algorithm, we used 256 5' 4-mer end motifs to identify distinct types of cfDNA cleavage patterns, referred to as "founder" end-motif profiles (F-profiles). F-profiles were associated with different DNA nucleases based on whether such patterns were disrupted in nuclease-knockout mouse models. Contributions of individual F-profiles in a cfDNA sample could be determined by deconvolutional analysis. We analyzed 93 murine cfDNA samples of different nuclease-deficient mice and identified six types of F-profiles. F-profiles I, II, and III were linked to deoxyribonuclease 1 like 3 (DNASE1L3), deoxyribonuclease 1 (DNASE1), and DNA fragmentation factor subunit beta (DFFB), respectively. We revealed that 42.9% of plasma cfDNA molecules were attributed to DNASE1L3-mediated fragmentation, whereas 43.4% of urinary cfDNA molecules involved DNASE1-mediated fragmentation. We further demonstrated that the relative contributions of F-profiles were useful to inform pathological states, such as autoimmune disorders and cancer. Among the six F-profiles, the use of F-profile I could inform the human patients with systemic lupus erythematosus. F-profile VI could be used to detect individuals with hepatocellular carcinoma, with an area under the receiver operating characteristic curve of 0.97. F-profile VI was more prominent in patients with nasopharyngeal carcinoma undergoing chemoradiotherapy. We proposed that this profile might be related to oxidative stress.


Assuntos
Ácidos Nucleicos Livres , Humanos , Camundongos , Animais , Ácidos Nucleicos Livres/genética , Desoxirribonucleases/genética , Camundongos Knockout , Endonucleases/genética , Fragmentação do DNA , Endodesoxirribonucleases/genética
2.
Clin Chem ; 69(2): 189-201, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36576350

RESUMO

BACKGROUND: Nuclear-derived cell-free DNA (cfDNA) molecules in blood plasma are nonrandomly fragmented, bearing a wealth of information related to tissues of origin. DNASE1L3 (deoxyribonuclease 1 like 3) is an important player in shaping the fragmentation of nuclear-derived cfDNA molecules, preferentially generating molecules with 5 CC dinucleotide termini (i.e., 5 CC-end motif). However, the fragment end properties of microbial cfDNA and its clinical implication remain to be explored. METHODS: We performed end motif analysis on microbial cfDNA fragments in plasma samples from patients with sepsis. A sequence context-based normalization method was used to minimize the potential biases for end motif analysis. RESULTS: The end motif profiles of microbial cfDNA appeared to resemble that of nuclear cfDNA (Spearman correlation coefficient: 0.82, P value 0.001). The CC-end motif was the most preferred end motif in microbial cfDNA, suggesting that DNASE1L3 might also play a role in the fragmentation of microbe-derived cfDNA in plasma. Of note, differential end motifs were present between microbial cfDNA originating from infection-causing pathogens (enriched at the CC-end) and contaminating microbial DNA potentially derived from reagents or the environment (nearly random). The use of fragment end signatures allowed differentiation between confirmed pathogens and contaminating microbes, with an area under the receiver operating characteristic curve of 0.99. The performance appeared to be superior to conventional analysis based on microbial cfDNA abundance alone. CONCLUSIONS: The use of fragmentomic features could facilitate the differentiation of underlying contaminating microbes from true pathogens in sepsis. This work demonstrates the potential usefulness of microbial cfDNA fragmentomics in metagenomics analysis.


Assuntos
Ácidos Nucleicos Livres , Sepse , Humanos , DNA/genética , Sepse/diagnóstico , Fragmentação do DNA
3.
Proc Natl Acad Sci U S A ; 119(44): e2209852119, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36288287

RESUMO

Cell-free DNA (cfDNA) fragmentation patterns contain important molecular information linked to tissues of origin. We explored the possibility of using fragmentation patterns to predict cytosine-phosphate-guanine (CpG) methylation of cfDNA, obviating the use of bisulfite treatment and associated risks of DNA degradation. This study investigated the cfDNA cleavage profile surrounding a CpG (i.e., within an 11-nucleotide [nt] window) to analyze cfDNA methylation. The cfDNA cleavage proportion across positions within the window appeared nonrandom and exhibited correlation with methylation status. The mean cleavage proportion was ∼twofold higher at the cytosine of methylated CpGs than unmethylated ones in healthy controls. In contrast, the mean cleavage proportion rapidly decreased at the 1-nt position immediately preceding methylated CpGs. Such differential cleavages resulted in a characteristic change in relative presentations of CGN and NCG motifs at 5' ends, where N represented any nucleotide. CGN/NCG motif ratios were correlated with methylation levels at tissue-specific methylated CpGs (e.g., placenta or liver) (Pearson's absolute r > 0.86). cfDNA cleavage profiles were thus informative for cfDNA methylation and tissue-of-origin analyses. Using CG-containing end motifs, we achieved an area under a receiver operating characteristic curve (AUC) of 0.98 in differentiating patients with and without hepatocellular carcinoma and enhanced the positive predictive value of nasopharyngeal carcinoma screening (from 19.6 to 26.8%). Furthermore, we elucidated the feasibility of using cfDNA cleavage patterns to deduce CpG methylation at single CpG resolution using a deep learning algorithm and achieved an AUC of 0.93. FRAGmentomics-based Methylation Analysis (FRAGMA) presents many possibilities for noninvasive prenatal, cancer, and organ transplantation assessment.


Assuntos
Ácidos Nucleicos Livres , Neoplasias Hepáticas , Gravidez , Feminino , Humanos , Ácidos Nucleicos Livres/genética , Biomarcadores Tumorais/genética , Metilação de DNA , Neoplasias Hepáticas/genética , Epigênese Genética , DNA/genética , Citosina , Guanina , Nucleotídeos , Fosfatos
4.
PLoS Genet ; 18(7): e1010262, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35793278

RESUMO

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.


Assuntos
Ácidos Nucleicos Livres , Neoplasias da Bexiga Urinária , Animais , Ácidos Nucleicos Livres/genética , DNA/genética , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Endodesoxirribonucleases/genética , Endonucleases , Humanos , Camundongos , Camundongos Knockout , Neoplasias da Bexiga Urinária/genética
5.
NPJ Genom Med ; 7(1): 14, 2022 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-35197474

RESUMO

Single-stranded ends of double-stranded DNA (jagged ends) are more abundant in urinary DNA than in plasma DNA. However, the lengths of jagged ends in urinary DNA remained undetermined, as a previous method used for urinary DNA jagged end sequencing analysis (Jag-seq) relied on unmethylation at CpG sites, limiting the resolution. Here, we performed high-resolution Jag-seq analysis using methylation at non-CpG cytosine sites, allowing determination of exact length of jagged ends. The urinary DNA bore longer jagged ends (~26-nt) than plasma DNA (~17-nt). The jagged end length distribution displayed 10-nt periodicities in urinary DNA, which were much less observable in plasma DNA. Amplitude of the 10-nt periodicities increased in patients with renal cell carcinoma. Heparin treatment of urine diminished the 10-nt periodicities. The urinary DNA jagged ends often extended into nucleosomal cores, suggesting potential interactions with histones. This study has thus advanced our knowledge of jagged ends in urine DNA.

6.
Prenat Diagn ; 41(3): 368-375, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33140416

RESUMO

OBJECTIVES: Due to the maternally-inherited nature of mitochondrial DNA (mtDNA), there is a lack of information regarding fetal mtDNA in the plasma of pregnant women. We aim to explore the presence and topologic forms of circulating fetal and maternal mtDNA molecules in surrogate pregnancies. METHODS: Genotypic differences between fetal and surrogate maternal mtDNA were used to identify the fetal and maternal mtDNA molecules in plasma. Plasma samples were obtained from the surrogate pregnant mothers. Using cleavage-end signatures of BfaI restriction enzyme, linear and circular mtDNA molecules in maternal plasma could be differentiated. RESULTS: Fetal-derived mtDNA molecules were mainly linear (median: 88%; range: 80%-96%), whereas approximately half of the maternal-derived mtDNA molecules were circular (median: 51%; range: 42%-60%). The fetal DNA fraction of linear mtDNA was lower (median absolute difference: 9.8%; range: 1.1%-27%) than that of nuclear DNA (median: 20%; range: 9.7%-35%). The fetal-derived linear mtDNA molecules were shorter than the maternal-derived ones. CONCLUSION: Fetal mtDNA is present in maternal plasma, and consists mainly of linear molecules. Surrogate pregnancies represent a valuable clinical scenario for exploring the biology and potential clinical applications of circulating mtDNA, for example, for pregnancies conceived following mitochondrial replacement therapy.


Assuntos
DNA Mitocondrial/genética , Feto/anormalidades , Mães Substitutas/estatística & dados numéricos , Adulto , DNA Mitocondrial/sangue , Feminino , Feto/fisiopatologia , Humanos , Herança Materna/genética , Moscou/epidemiologia , Plasma/microbiologia , Gravidez
7.
Clin Chem ; 65(9): 1161-1170, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31263037

RESUMO

BACKGROUND: Cellular mitochondrial DNA (mtDNA) is organized as circular, covalently closed and double-stranded DNA. Studies have demonstrated the presence of short mtDNA fragments in plasma. It is not known whether circular mtDNA might concurrently exist with linear mtDNA in plasma. METHODS: We elucidated the topology of plasma mtDNA using restriction enzyme BfaI cleavage signatures on mtDNA fragment ends to differentiate linear and circular mtDNA. mtDNA fragments with both ends carrying BfaI cleavage signatures were defined as circular-derived mtDNA, whereas those with no cleavage signature or with 1 cleavage signature were defined as linear-derived mtDNA. An independent assay using exonuclease V to remove linear DNA followed by restriction enzyme MspI digestion was used for confirming the conclusions based on BfaI cleavage analysis. We analyzed the presence of BfaI cleavage signatures on plasma DNA ends in nonhematopoietically and hematopoietically derived DNA molecules by sequencing plasma DNA of patients with liver transplantation and bone marrow transplantation. RESULTS: Both linear and circular mtDNA coexisted in plasma. In patients with liver transplantation, donor-derived (i.e., liver) mtDNA molecules were mainly linear (median fraction, 91%; range, 75%-97%), whereas recipient-derived (i.e., hematopoietic) mtDNA molecules were mainly circular (median fraction, 88%; range, 77%-93%). The proportion of linear mtDNA was well correlated with liver DNA contribution in the plasma DNA pool (r = 0.83; P value = 0.0008). Consistent data were obtained from a bone marrow transplantation recipient in whom the donor-derived (i.e., hematopoietic) mtDNA molecules were predominantly circular. CONCLUSIONS: Linear and circular mtDNA molecules coexist in plasma and may have different tissue origins.


Assuntos
DNA Mitocondrial/sangue , Adulto , Transplante de Medula Óssea , DNA Mitocondrial/química , DNA Mitocondrial/genética , Desoxirribonucleases de Sítio Específico do Tipo II/química , Feminino , Humanos , Transplante de Fígado , Masculino , Conformação de Ácido Nucleico , Gravidez
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