RESUMO
Recent studies have revealed an unexplored population of long cell-free DNA (cfDNA) molecules in human plasma using long-read sequencing technologies. However, the biological properties of long cfDNA molecules (>500 bp) remain largely unknown. To this end, we have investigated the origins of long cfDNA molecules from different genomic elements. Analysis of plasma cfDNA using long-read sequencing reveals an uneven distribution of long molecules from across the genome. Long cfDNA molecules show overrepresentation in euchromatic regions of the genome, in sharp contrast to short DNA molecules. We observe a stronger relationship between the abundance of long molecules and mRNA gene expression levels, compared with short molecules (Pearson's r = 0.71 vs. -0.14). Moreover, long and short molecules show distinct fragmentation patterns surrounding CpG sites. Leveraging the cleavage preferences surrounding CpG sites, the combined cleavage ratios of long and short molecules can differentiate patients with hepatocellular carcinoma (HCC) from non-HCC subjects (AUC = 0.87). We also investigated knockout mice in which selected nuclease genes had been inactivated in comparison with wild-type mice. The proportion of long molecules originating from transcription start sites are lower in Dffb-deficient mice but higher in Dnase1l3-deficient mice compared with that of wild-type mice. This work thus provides new insights into the biological properties and potential clinical applications of long cfDNA molecules.
Assuntos
Carcinoma Hepatocelular , Ácidos Nucleicos Livres , Neoplasias Hepáticas , Humanos , Animais , Camundongos , Ácidos Nucleicos Livres/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , DNA/genética , Genômica , Camundongos Knockout , Endodesoxirribonucleases/genéticaRESUMO
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/genéticaRESUMO
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 â¼twofold 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 , FosfatosRESUMO
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/genéticaRESUMO
Cell-free DNA (cfDNA) is a widely used noninvasive biomarker for diagnosis and prognosis of multiple disease states. Emerging evidence suggests that cfDNA might not just be passive waste products of cell death but could have a physiological and pathological function in inflammation and autoimmunity. The balance of cfDNA generation and clearance may thus be vital in health and disease. In particular, plasma nuclease activity has been linked to multiple pathologies including cancer and systemic lupus erythematosus (SLE) and associated with profound changes in the nonrandom fragmentation of cfDNA. Lastly, in this review, we explore the effects of DNA fragmentation factor B (DFFB), DNASE1L3, and DNASE1 on cfDNA levels and their fragmentomic profiles, and what these recent insights reveal about the biology of cfDNA.
Assuntos
Ácidos Nucleicos Livres/genética , Desoxirribonuclease I/genética , Desoxirribonucleases/genética , Endodesoxirribonucleases/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Autoimunidade/genética , Ácidos Nucleicos Livres/sangue , Fragmentação do DNA , Desoxirribonuclease I/sangue , Desoxirribonucleases/sangue , Endodesoxirribonucleases/sangue , Humanos , Inflamação/sangue , Inflamação/genética , Inflamação/patologia , Proteínas de Ligação a Poli-ADP-Ribose/sangueRESUMO
The effects of DNASE1L3 or DNASE1 deficiency on cell-free DNA (cfDNA) methylation were explored in plasma of mice deficient in these nucleases and in DNASE1L3-deficient humans. Compared to wild-type cfDNA, cfDNA in DNASE1L3-deficient mice was significantly hypomethylated, while cfDNA in DNASE1-deficient mice was hypermethylated. The cfDNA hypomethylation in DNASE1L3-deficient mice was due to increased fragmentation and representation from open chromatin regions (OCRs) and CpG islands (CGIs). These findings were absent in DNASE1-deficient mice, demonstrating the preference of DNASE1 to cleave in hypomethylated OCRs and CGIs. We also observed a substantial decrease of fragment ends at methylated CpGs in the absence of DNASE1L3, thereby demonstrating that DNASE1L3 prefers to cleave at methylated CpGs. Furthermore, we found that methylation levels of cfDNA varied by fragment size in a periodic pattern, with cfDNA of specific sizes being more hypomethylated and enriched for OCRs and CGIs. These findings were confirmed in DNASE1L3-deficient human cfDNA. Thus, we have found that nuclease-mediated cfDNA fragmentation markedly affects cfDNA methylation level on a genome-wide scale. This work provides a foundational understanding of the relationship between methylation, nuclease biology, and cfDNA fragmentation.
Assuntos
Ácidos Nucleicos Livres , Fragmentação do DNA , Endodesoxirribonucleases , Animais , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/metabolismo , Cromatina , Ilhas de CpG/genética , Metilação de DNA , Endodesoxirribonucleases/genética , Humanos , CamundongosRESUMO
BACKGROUND: The analysis of haplotypes of variants is important for pharmacogenomics analysis and noninvasive prenatal testing for monogenic diseases. However, there is a lack of robust methods for targeted haplotyping. METHODS: We developed digital PCR haplotype sequencing (dHapSeq) for targeted haplotyping of variants, which is a method that compartmentalizes long DNA molecules into droplets. Within one droplet, 2 target regions are PCR amplified from one template molecule, and their amplicons are fused together. The fused products are then sequenced to determine the phase relationship of the single nucleotide polymorphism (SNP) alleles. The entire haplotype of 10s of SNPs can be deduced after the phase relationship of individual SNPs are determined in a pairwise manner. We applied dHapSeq to noninvasive prenatal testing in 4 families at risk for thalassemia and utilized it to detect NUDT15 diplotypes for predicting drug tolerance in pediatric acute lymphoblastic leukemia (72 cases and 506 controls). RESULTS: For SNPs within 40â kb, phase relation can be determined with 100% accuracy. In 7 trio families, the haplotyping results for 97 SNPs spanning 185â kb determined by dHapSeq were concordant with the results deduced from the genotypes of both parents and the fetus. In 4 thalassemia families, a 19.3-kb Southeast Asian deletion was successfully phased with 97 downstream SNPs, enabling noninvasive determination of fetal inheritance using relative haplotype dosage analysis. In the NUDT15 analysis, the variant status and phase of the variants were successfully determined in all cases and controls. CONCLUSIONS: The dHapSeq represents a robust and scalable haplotyping approach with numerous clinical and research applications.
Assuntos
Haplótipos , Teste Pré-Natal não Invasivo , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Humanos , Reação em Cadeia da Polimerase/métodos , Feminino , Teste Pré-Natal não Invasivo/métodos , Gravidez , Testes Farmacogenômicos/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Análise de Sequência de DNA/métodos , Talassemia/genética , Talassemia/diagnósticoRESUMO
In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g., <500 bp). The existence of long cell-free DNA molecules has been poorly explored. In this study, we demonstrated that single-molecule real-time sequencing allowed us to detect and analyze a substantial proportion of long DNA molecules from both fetal and maternal sources in maternal plasma. Such molecules were beyond the size detection limits of short-read sequencing technologies. The proportions of long cell-free DNA molecules in maternal plasma over 500 bp were 15.5%, 19.8%, and 32.3% for the first, second, and third trimesters, respectively. The longest fetal-derived plasma DNA molecule observed was 23,635 bp. Long plasma DNA molecules demonstrated predominance of A or G 5' fragment ends. Pregnancies with preeclampsia demonstrated a reduction in long maternal plasma DNA molecules, reduced frequencies for selected 5' 4-mer end motifs ending with G or A, and increased frequencies for selected motifs ending with T or C. Finally, we have developed an approach that employs the analysis of methylation patterns of the series of CpG sites on a long DNA molecule for determining its tissue origin. This approach achieved an area under the curve of 0.88 in differentiating between fetal and maternal plasma DNA molecules, enabling the determination of maternal inheritance and recombination events in the fetal genome. This work opens up potential clinical utilities of long cell-free DNA analysis in maternal plasma including noninvasive prenatal testing of monogenic diseases and detection/monitoring of pregnancy-associated disorders such as preeclampsia.
Assuntos
Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Adulto , Cromossomos/genética , Simulação por Computador , Feminino , Feto , Humanos , Gravidez , Imagem Individual de MoléculaRESUMO
5-Methylcytosine (5mC) is an important type of epigenetic modification. Bisulfite sequencing (BS-seq) has limitations, such as severe DNA degradation. Using single molecule real-time sequencing, we developed a methodology to directly examine 5mC. This approach holistically examined kinetic signals of a DNA polymerase (including interpulse duration and pulse width) and sequence context for every nucleotide within a measurement window, termed the holistic kinetic (HK) model. The measurement window of each analyzed double-stranded DNA molecule comprised 21 nucleotides with a cytosine in a CpG site in the center. We used amplified DNA (unmethylated) and M.SssI-treated DNA (methylated) (M.SssI being a CpG methyltransferase) to train a convolutional neural network. The area under the curve for differentiating methylation states using such samples was up to 0.97. The sensitivity and specificity for genome-wide 5mC detection at single-base resolution reached 90% and 94%, respectively. The HK model was then tested on human-mouse hybrid fragments in which each member of the hybrid had a different methylation status. The model was also tested on human genomic DNA molecules extracted from various biological samples, such as buffy coat, placental, and tumoral tissues. The overall methylation levels deduced by the HK model were well correlated with those by BS-seq (r = 0.99; P < 0.0001) and allowed the measurement of allele-specific methylation patterns in imprinted genes. Taken together, this methodology has provided a system for simultaneous genome-wide genetic and epigenetic analyses.
Assuntos
Citosina/metabolismo , Metilação de DNA/genética , Análise de Sequência de DNA , Imagem Individual de Molécula , Animais , Sequência de Bases , DNA/metabolismo , Impressão Genômica , Humanos , Camundongos , Modelos BiológicosRESUMO
Cell-free DNA (cf.DNA) is a powerful noninvasive biomarker for cancer and prenatal testing, and it circulates in plasma as short fragments. To elucidate the biology of cf.DNA fragmentation, we explored the roles of deoxyribonuclease 1 (DNASE1), deoxyribonuclease 1 like 3 (DNASE1L3), and DNA fragmentation factor subunit beta (DFFB) with mice deficient in each of these nucleases. By analyzing the ends of cf.DNA fragments in each type of nuclease-deficient mice with those in wild-type mice, we show that each nuclease has a specific cutting preference that reveals the stepwise process of cf.DNA fragmentation. Essentially, we demonstrate that cf.DNA is generated first intracellularly with DFFB, intracellular DNASE1L3, and other nucleases. Then, cf.DNA fragmentation continues extracellularly with circulating DNASE1L3 and DNASE1. With the use of heparin to disrupt the nucleosomal structure, we also show that the 10 bp periodicity originates from the cutting of DNA within an intact nucleosomal structure. Altogether, this work establishes a model of cf.DNA fragmentation.
Assuntos
Ácidos Nucleicos Livres/metabolismo , Cromatina/metabolismo , Fragmentação do DNA , Desoxirribonuclease I/fisiologia , Desoxirribonucleases/fisiologia , Endodesoxirribonucleases/fisiologia , Nucleossomos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , Animais , Ácidos Nucleicos Livres/genética , Cromatina/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Nucleossomos/genéticaRESUMO
Plasma DNA fragmentomics is an emerging area in cell-free DNA diagnostics and research. In murine models, it has been shown that the extracellular DNase, DNASE1L3, plays a role in the fragmentation of plasma DNA. In humans, DNASE1L3 deficiency causes familial monogenic systemic lupus erythematosus with childhood onset and anti-dsDNA reactivity. In this study, we found that human patients with DNASE1L3 disease-associated gene variations showed aberrations in size and a reduction of a "CC" end motif of plasma DNA. Furthermore, we demonstrated that DNA from DNASE1L3-digested cell nuclei showed a median length of 153 bp with CC motif frequencies resembling plasma DNA from healthy individuals. Adeno-associated virus-based transduction of Dnase1l3 into Dnase1l3-deficient mice restored the end motif profiles to those seen in the plasma DNA of wild-type mice. Our findings demonstrate that DNASE1L3 is an important player in the fragmentation of plasma DNA, which appears to act in a cell-extrinsic manner to regulate plasma DNA size and motif frequency.
Assuntos
DNA/genética , Endodesoxirribonucleases/genética , Lúpus Eritematoso Sistêmico/genética , Mutação , Animais , Estudos de Casos e Controles , DNA/sangue , Fragmentação do DNA , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animais de Doenças , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/metabolismo , Terapia Genética , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Lúpus Eritematoso Sistêmico/enzimologia , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Camundongos Transgênicos , Especificidade por Substrato , Transdução GenéticaRESUMO
Cell-free DNA in plasma has been used for noninvasive prenatal testing and cancer liquid biopsy. The physical properties of cell-free DNA fragments in plasma, such as fragment sizes and ends, have attracted much recent interest, leading to the emerging field of cell-free DNA fragmentomics. However, one aspect of plasma DNA fragmentomics as to whether double-stranded plasma molecules might carry single-stranded ends, termed a jagged end in this study, remains underexplored. We have developed two approaches for investigating the presence of jagged ends in a plasma DNA pool. These approaches utilized DNA end repair to introduce differential methylation signals between the original sequence and the jagged ends, depending on whether unmethylated or methylated cytosines were used in the DNA end-repair procedure. The majority of plasma DNA molecules (87.8%) were found to bear jagged ends. The jaggedness varied according to plasma DNA fragment sizes and appeared to be in association with nucleosomal patterns. In the plasma of pregnant women, the jaggedness of fetal DNA molecules was higher than that of the maternal counterparts. The jaggedness of plasma DNA correlated with the fetal DNA fraction. Similarly, in the plasma of cancer patients, tumor-derived DNA molecules in patients with hepatocellular carcinoma showed an elevated jaggedness compared with nontumoral DNA. In mouse models, knocking out of the Dnase1 gene reduced jaggedness, whereas knocking out of the Dnase1l3 gene enhanced jaggedness. Hence, plasma DNA jagged ends represent an intrinsic property of plasma DNA and provide a link between nuclease activities and the fragmentation of plasma DNA.
Assuntos
Ácidos Nucleicos Livres/sangue , Fragmentação do DNA , Metilação de DNA/genética , DNA/sangue , DNA/genética , Animais , Carcinoma Hepatocelular/genética , Ácidos Nucleicos Livres/genética , Reparo do DNA por Junção de Extremidades/genética , Endodesoxirribonucleases/genética , Feminino , Humanos , Neoplasias Hepáticas/genética , Camundongos , Camundongos Knockout , Nucleossomos/genética , GravidezRESUMO
BACKGROUND: Recent studies using single molecule, real-time (SMRT) sequencing revealed a substantial population of analyzable long cell-free DNA (cfDNA) in plasma. Potential clinical utilities of such long cfDNA in pregnancy and cancer have been demonstrated. However, the performance of different long-read sequencing platforms for the analysis of long cfDNA remains unknown. METHODS: Size biases of SMRT sequencing by Pacific Biosciences (PacBio) and nanopore sequencing by Oxford Nanopore Technologies (ONT) were evaluated using artificial mixtures of sonicated human and mouse DNA of different sizes. cfDNA from plasma samples of pregnant women at different trimesters, hepatitis B carriers, and patients with hepatocellular carcinoma were sequenced with the 2 platforms. RESULTS: Both platforms showed biases to sequence longer (1500 bp vs 200 bp) DNA fragments, with PacBio showing a stronger bias (5-fold overrepresentation of long fragments vs 2-fold in ONT). Percentages of cfDNA fragments 500 bp were around 6-fold higher in PacBio compared with ONT. End motif profiles of cfDNA from PacBio and ONT were similar, yet exhibited platform-dependent patterns. Tissue-of-origin analysis based on single-molecule methylation patterns showed comparable performance on both platforms. CONCLUSIONS: SMRT sequencing generated data with higher percentages of long cfDNA compared with nanopore sequencing. Yet, a higher number of long cfDNA fragments eligible for the tissue-of-origin analysis could be obtained from nanopore sequencing due to its much higher throughput. When analyzing the size and end motif of cfDNA, one should be aware of the analytical characteristics and possible biases of the sequencing platforms being used.
Assuntos
Ácidos Nucleicos Livres , Neoplasias Hepáticas , Sequenciamento por Nanoporos , Humanos , Feminino , Gravidez , Animais , Camundongos , Ácidos Nucleicos Livres/genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , DNA/genéticaRESUMO
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 DNARESUMO
OBJECTIVE: Long cell-free DNA (cfDNA) can be found in the plasma of pregnant women and cancer patients. We investigated if droplet digital PCR (ddPCR) can analyze such molecules for diagnostic purposes using preeclampsia as a model. METHOD: Plasma samples from ten preeclamptic and sixteen normal pregnancies were analyzed. Two ddPCR assays targeting a single-copy gene, VCP, and one ddPCR assay targeting LINE-1 repetitive regions were used to measure the percentages of long cfDNA >533, 1001, and 170 bp, respectively. The LINE-1 assay was developed as guided by in silico PCR analyses to better differentiate preeclamptic and normal pregnancies. RESULTS: Preeclamptic patients had a significantly lower median percentage of long cfDNA than healthy pregnant controls, as determined by the LINE-1 170 bp assay (28.9% vs. 35.1%, p < 0.0001) and the VCP 533 bp assay (6.6% vs. 8.7%, p = 0.014). The LINE-1 assay provided a better differentiation than the VCP 533 bp assay (area under ROC curves, 0.94 vs. 0.79). CONCLUSION: ddPCR is a cost-effective approach for unlocking diagnostic information carried by long cfDNA in plasma and may have applications for the detection of preeclampsia. Further longitudinal studies with larger cohorts are required to assess the clinical utility of this test.
RESUMO
We explored the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. Through sequencing following either restriction enzyme or Tn5 transposase treatment, we identified eccDNA molecules in the plasma of pregnant women. These eccDNA molecules showed bimodal size distributions peaking at â¼202 and â¼338 bp with distinct 10-bp periodicity observed throughout the size ranges within both peaks, suggestive of their nucleosomal origin. Also, the predominance of the 338-bp peak of eccDNA indicated that eccDNA had a larger size distribution than linear DNA in human plasma. Moreover, eccDNA of fetal origin were shorter than the maternal eccDNA. Genomic annotation of the overall population of eccDNA molecules revealed a preference of these molecules to be generated from 5'-untranslated regions (5'-UTRs), exonic regions, and CpG island regions. Two sets of trinucleotide repeat motifs flanking the junctional sites of eccDNA supported multiple possible models for eccDNA generation. This work highlights the topologic analysis of plasma DNA, which is an emerging direction for circulating nucleic acid research and applications.
Assuntos
Ácidos Nucleicos Livres/isolamento & purificação , DNA Circular/isolamento & purificação , Plasma/química , Ácidos Nucleicos Livres/química , Ácidos Nucleicos Livres/genética , DNA Circular/química , DNA Circular/genética , Feminino , Genoma Humano , Hong Kong , Humanos , Teste Pré-Natal não Invasivo , GravidezRESUMO
BACKGROUND: Quantitative measurement of plasma Epstein-Barr virus (EBV) DNA by real-time PCR at the end of primary treatment is a robust prognostic marker for nasopharyngeal carcinoma (NPC) patients. However, up to 40% of patients who would later develop disease recurrence had undetectable post-treatment plasma EBV DNA. Targeted sequencing for the entire EBV genome potentially allows a more comprehensive and unbiased detection of plasma EBV DNA and enables the use of other parameters such as fragment size as biomarkers. Hence, we explored if plasma EBV DNA sequencing might allow more accurate prognostication of NPC patients. PATIENTS AND METHODS: Plasma samples collected from 769 patients with stage IIB-IVB NPC at 6-8 weeks after radiotherapy were analysed using targeted sequencing for EBV DNA. RESULTS: The sensitivities of the PCR-based analysis, at a cut-off of any detectable levels of plasma EBV DNA, for prediction of local and distant recurrences were 42.3% and 85.3%, respectively. The sequencing-based analysis (involving quantitation and size profiling) achieved better performance for both local and distant recurrences than PCR. Using a cut-off of the proportion of plasma EBV DNA deduced by sequencing at 0.01%, the sensitivities of the sequencing-based analysis for local and distant recurrences were 88.5% and 97.1%, with the resultant negative predictive values of 99.1% and 99.4%, respectively. Among patients with undetectable EBV DNA on quantitative PCR, sequencing could further define a subgroup that enjoyed superior survival outcomes based on the proportion of plasma EBV DNA, with a 5-year progression-free survival (PFS) approaching 90%. On multivariate analysis, sequencing-based quantitative level of plasma EBV DNA was the independent prognostic factor with the highest hazard ratio for prediction of overall survival and PFS. CONCLUSION: NPC prognostication using post-treatment plasma EBV DNA could be enhanced through sequencing.
Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias Nasofaríngeas , DNA Viral/genética , Herpesvirus Humano 4/genética , Humanos , Carcinoma Nasofaríngeo/patologia , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/terapia , Recidiva Local de Neoplasia/genética , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Medição de RiscoRESUMO
Cell-free DNA (cfDNA) in human plasma is a class of biomarkers with many current and potential future diagnostic applications. Recent studies have shown that cfDNA molecules are not randomly fragmented and possess information related to their tissues of origin. Pathologies causing death of cells from particular tissues result in perturbations in the relative distribution of DNA from the affected tissues. Such tissue-of-origin analysis is particularly useful in the development of liquid biopsies for cancer. It is therefore of value to accurately determine the relative contributions of the tissues to the plasma DNA pool in a simultaneous manner. In this work, we report that in open chromatin regions, cfDNA molecules show characteristic fragmentation patterns reflected by sequencing coverage imbalance and differentially phased fragment end signals. The latter refers to differences in the read densities of sequences corresponding to the orientation of the upstream and downstream ends of cfDNA molecules in relation to the reference genome. Such cfDNA fragmentation patterns preferentially occur in tissue-specific open chromatin regions where the corresponding tissues contributed DNA into the plasma. Quantitative analyses of such signals allow measurement of the relative contributions of various tissues toward the plasma DNA pool. These findings were validated by plasma DNA sequencing data obtained from pregnant women, organ transplantation recipients, and cancer patients. Orientation-aware plasma DNA fragmentation analysis therefore has potential diagnostic applications in noninvasive prenatal testing, organ transplantation monitoring, and cancer liquid biopsy.
Assuntos
Biomarcadores Tumorais/sangue , Ácidos Nucleicos Livres/genética , Cromatina/genética , Fragmentação do DNA , Biomarcadores Tumorais/normas , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/química , Cromatina/química , Humanos , Especificidade de Órgãos , Padrões de ReferênciaRESUMO
BACKGROUND: Jagged ends of plasma DNA are a recently recognized class of fragmentomic markers for cell-free DNA, reflecting the activity of nucleases. A number of recent studies have also highlighted the importance of jagged ends in the context of pregnancy and oncology. However, knowledge regarding the generation of jagged ends is incomplete. METHODS: Jaggedness of plasma DNA was analyzed based on Jag-seq, which utilized the differential methylation signals introduced by the DNA end-repair process. We investigated the jagged ends in plasma DNA using mouse models by deleting the deoxyribonuclease 1 (Dnase1), DNA fragmentation factor subunit beta (Dffb), or deoxyribonuclease 1 like 3 (Dnase1l3) gene. RESULTS: Aberrations in the profile of plasma DNA jagged ends correlated with the type of nuclease that had been genetically deleted, depending on nucleosomal structures. The deletion of Dnase1l3 led to a significant reduction of jaggedness for those plasma DNA molecules involving more than 1 nucleosome (e.g., size ranges 240-290â bp, 330-380â bp, and 420-470â bp). However, less significant effects of Dnase1 and Dffb deletions were observed regarding different sizes of DNA fragments. Interestingly, the aberration in plasma DNA jagged ends related to multinucleosomes was observed in human subjects with familial systemic lupus erythematosus with Dnase1l3 deficiency and human subjects with sporadic systemic lupus erythematosus. CONCLUSIONS: Detailed understanding of the relationship between nuclease and plasma DNA jaggedness has opened up avenues for biomarker development.