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
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
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: 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
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.
Assuntos
Ácidos Nucleicos Livres , Lúpus Eritematoso Sistêmico , Animais , Biomarcadores , Ácidos Nucleicos Livres/genética , DNA/genética , Desoxirribonucleases/genética , Endodesoxirribonucleases/genética , Feminino , Humanos , Lúpus Eritematoso Sistêmico/genética , Camundongos , Nucleossomos/genética , GravidezRESUMO
Circulating DNA in plasma consists of short DNA fragments. The biological processes generating such fragments are not well understood. DNASE1L3 is a secreted DNASE1-like nuclease capable of digesting DNA in chromatin, and its absence causes anti-DNA responses and autoimmunity in humans and mice. We found that the deletion of Dnase1l3 in mice resulted in aberrations in the fragmentation of plasma DNA. Such aberrations included an increase in short DNA molecules below 120 bp, which was positively correlated with anti-DNA antibody levels. We also observed an increase in long, multinucleosomal DNA molecules and decreased frequencies of the most common end motifs found in plasma DNA. These aberrations were independent of anti-DNA response, suggesting that they represented a primary effect of DNASE1L3 loss. Pregnant Dnase1l3-/- mice carrying Dnase1l3+/- fetuses showed a partial restoration of normal frequencies of plasma DNA end motifs, suggesting that DNASE1L3 from Dnase1l3-proficient fetuses could enter maternal systemic circulation and affect both fetal and maternal DNA fragmentation in a systemic as well as local manner. However, the observed shortening of circulating fetal DNA relative to maternal DNA was not affected by the deletion of Dnase1l3 Collectively, our findings demonstrate that DNASE1L3 plays a role in circulating plasma DNA homeostasis by enhancing fragmentation and influencing end-motif frequencies. These results support a distinct role of DNASE1L3 as a regulator of the physical form and availability of cell-free DNA and may have important implications for the mechanism whereby this enzyme prevents autoimmunity.
Assuntos
Ácidos Nucleicos Livres/sangue , Fragmentação do DNA , DNA/sangue , Endodesoxirribonucleases/metabolismo , Motivos de Nucleotídeos , Animais , Ácidos Nucleicos Livres/genética , DNA/genética , Endodesoxirribonucleases/genética , Feminino , Feto/metabolismo , Deleção de Genes , Camundongos , Camundongos Knockout , GravidezRESUMO
Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma.
Assuntos
Carcinoma Hepatocelular/genética , Metilação de DNA , DNA/genética , Neoplasias Hepáticas/genética , Análise de Sequência de DNA/métodos , Transplante de Tecidos , Adulto , Algoritmos , Linfócitos B/metabolismo , Transplante de Medula Óssea , Carcinoma Hepatocelular/sangue , DNA/sangue , DNA/química , Variações do Número de Cópias de DNA/genética , Feminino , Feto/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/sangue , Transplante de Fígado , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Placenta/metabolismo , Gravidez , Linfócitos T/metabolismoRESUMO
BACKGROUND: There is much interest in the tissue of origin of circulating DNA in plasma. Data generated using DNA methylation markers have suggested that hematopoietic cells of white cell lineages are important contributors to the circulating DNA pool. However, it is not known whether cells of the erythroid lineage would also release DNA into the plasma. METHODS: Using high-resolution methylation profiles of erythroblasts and other tissue types, 3 genomic loci were found to be hypomethylated in erythroblasts but hypermethylated in other cell types. We developed digital PCR assays for measuring erythroid DNA using the differentially methylated region for each locus. RESULTS: Based on the methylation marker in the ferrochelatase gene, erythroid DNA represented a median of 30.1% of the plasma DNA of healthy subjects. In subjects with anemia of different etiologies, quantitative analysis of circulating erythroid DNA could reflect the erythropoietic activity in the bone marrow. For patients with reduced erythropoietic activity, as exemplified by aplastic anemia, the percentage of circulating erythroid DNA was decreased. For patients with increased but ineffective erythropoiesis, as exemplified by ß-thalassemia major, the percentage was increased. In addition, the plasma concentration of erythroid DNA was found to correlate with treatment response in aplastic anemia and iron deficiency anemia. Plasma DNA analysis using digital PCR assays targeting the other 2 differentially methylated regions showed similar findings. CONCLUSIONS: Erythroid DNA is a hitherto unrecognized major component of the circulating DNA pool and is a noninvasive biomarker for differential diagnosis and monitoring of anemia.
Assuntos
Anemia/sangue , Anemia/genética , Metilação de DNA , DNA/sangue , DNA/genética , Eritroblastos/patologia , Anemia/diagnóstico , Anemia/patologia , Anemia Aplástica/sangue , Anemia Aplástica/diagnóstico , Anemia Aplástica/genética , Anemia Aplástica/patologia , Anemia Ferropriva/sangue , Anemia Ferropriva/diagnóstico , Anemia Ferropriva/genética , Anemia Ferropriva/patologia , Diagnóstico Diferencial , Eritroblastos/metabolismo , Eritropoese , Ferroquelatase/genética , Humanos , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Talassemia beta/sangue , Talassemia beta/diagnóstico , Talassemia beta/genética , Talassemia beta/patologiaRESUMO
We performed a high-resolution analysis of the biological characteristics of plasma DNA in systemic lupus erythematosus (SLE) patients using massively parallel genomic and methylomic sequencing. A number of plasma DNA abnormalities were found. First, aberrations in measured genomic representations (MGRs) were identified in the plasma DNA of SLE patients. The extent of the aberrations in MGRs correlated with anti-double-stranded DNA (anti-dsDNA) antibody level. Second, the plasma DNA of active SLE patients exhibited skewed molecular size-distribution profiles with a significantly increased proportion of short DNA fragments. The extent of plasma DNA shortening in SLE patients correlated with the SLE disease activity index (SLEDAI) and anti-dsDNA antibody level. Third, the plasma DNA of active SLE patients showed decreased methylation densities. The extent of hypomethylation correlated with SLEDAI and anti-dsDNA antibody level. To explore the impact of anti-dsDNA antibody on plasma DNA in SLE, a column-based protein G capture approach was used to fractionate the IgG-bound and non-IgG-bound DNA in plasma. Compared with healthy individuals, SLE patients had higher concentrations of IgG-bound DNA in plasma. More IgG binding occurs at genomic locations showing increased MGRs. Furthermore, the IgG-bound plasma DNA was shorter in size and more hypomethylated than the non-IgG-bound plasma DNA. These observations have enhanced our understanding of the spectrum of plasma DNA aberrations in SLE and may provide new molecular markers for SLE. Our results also suggest that caution should be exercised when interpreting plasma DNA-based noninvasive prenatal testing and cancer testing conducted for SLE patients.
Assuntos
Biomarcadores/sangue , Metilação de DNA , DNA/sangue , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/genética , Adulto , Idoso , Aberrações Cromossômicas , Ilhas de CpG , Epigênese Genética , Feminino , Regulação da Expressão Gênica , Biblioteca Gênica , Genoma Humano , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina G/análise , Pessoa de Meia-Idade , Análise de Sequência de DNARESUMO
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.
RESUMO
BACKGROUND: We hypothesized that liver-derived mRNA, such as ALB (albumin) mRNA, would be released into human plasma with liver cell death. METHODS: We genotyped ALB mRNA molecules in samples of plasma and whole blood from liver and bone marrow transplant recipients by RNA single-nucleotide polymorphism analysis. Plasma and whole blood ALB mRNA genotypes were compared with the DNA genotypes of the recipients and donors. A reverse-transcription quantitative real-time PCR assay was used to measure plasma ALB mRNA concentrations in 107 patients [hepatocellular carcinoma (HCC), cirrhosis, or chronic hepatitis B (CHB)] and 207 healthy controls. RESULTS: The RNA genotype data revealed ALB mRNA in plasma to be liver derived, whereas tissue compartments other than the liver also contributed to the ALB mRNA detected in whole blood. Statistically significant increases in plasma ALB mRNA concentrations were observed for HCC, cirrhosis, and active CHB, compared with controls. A cutoff of 835 copies/mL of plasma ALB mRNA identified by ROC curve analysis showed 85.5% diagnostic sensitivity and 92.8% diagnostic specificity for the detection of liver pathologies. Only 21.5% of patients with liver pathologies had increased alanine aminotransferase (ALT) activities, whereas 73.8% had increased plasma ALB mRNA concentrations. Only 48.6% of the HCC patients had increased serum alpha-fetoprotein concentrations, whereas 91.4% had increased plasma ALB mRNA concentrations. CONCLUSIONS: ALB mRNA is liver specific in plasma, but not in whole blood. Plasma ALB mRNA is increased in some liver pathologies and may be more diagnostically sensitive than alpha-fetoprotein and ALT.
Assuntos
Hepatopatias/metabolismo , Fígado/metabolismo , RNA Mensageiro/sangue , Albumina Sérica/genética , Adulto , Idoso , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Curva ROCRESUMO
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 tissue-of-origin information and represent a new class of biomarkers in the nascent field of fragmentomics.This article is highlighted in the In This Issue feature, p. 627.
Assuntos
DNA/sangue , Neoplasias Hepáticas/genética , Transplante de Fígado/métodos , Feminino , Humanos , GravidezRESUMO
BACKGROUND: The term "transrenal DNA" was coined in 2000 to signify that DNA in urine may come from the passage of plasma DNA through the kidney barrier. Although DNA in the urine has the potential to provide a completely noninvasive source of nucleic acids for molecular diagnosis, its existence remains controversial. METHODS: We obtained blood and urine samples from 22 hematopoietic stem cell transplant (HSCT) recipients and used fluorescence in situ hybridization, PCR for short tandem repeats, mass spectrometry, quantitative PCR, and immunofluorescence detection to study donor-derived DNA in the urine. RESULTS: All HSCT recipients exhibited high amounts of donor-derived DNA in buffy coat and plasma samples. Male donor-derived DNA was detected in supernatants of urine samples from all 5 female sex-mismatched HSCT recipients. Surprisingly, the amount of DNA in urine supernatants was not correlated with the plasma value. Moreover, cell-free urine supernatants contained DNA fragments >350 bp that were absent in plasma. Donor-derived polymorphs were detected in urine by fluorescence in situ hybridization. Coincidentally, donor-derived cytokeratin-producing epithelial cells were discovered in urine samples from 3 of 10 sex-mismatched HSCT recipients as long as 14.2 years after transplantation. CONCLUSIONS: This report is the first to demonstrate the presence of donor-derived DNA in the urine of HSCT recipients; however, we show that much of this DNA originates from donor-derived cells, rather than from the transrenal passage of cell-free plasma DNA. Our discovery of donor-derived cytokeratin-producing epithelial cells raises interesting biological and therapeutic implications, e.g., the capacity of marrow stem cells to serve as an extrarenal source for renal tubule regeneration.
Assuntos
DNA/urina , Transplante de Células-Tronco Hematopoéticas , Rim/fisiologia , Modelos Biológicos , Doadores de Tecidos , DNA/sangue , DNA/genética , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Transplante HomólogoRESUMO
OBJECTIVE: (i) To investigate the intra-rater, inter-rater and test-retest reliability and minimal detectable change of the Alternate Step Test (AST) when assessing people with chronic stroke. (ii) To quantify the correlation between AST times and stroke-specific impairments. DESIGN: Cross-sectional study. SETTING: University-based rehabilitation centre. PARTICIPANTS: A convenience sample of 86 participants: 45 with chronic stroke, and 41 healthy elderly subjects. METHODS: The AST was administered along with the Fugl-Meyer Lower Extremity Assessment (FMA-LE), the Five Times Sit-To-Stand Test (FTSTS), limits of stability (LOS) measurements, Berg Balance Scale (BBS) scores, Chinese-translated Activities-specific Balance Confidence Scale (ABC-C) ratings, and the Timed "Up and Go" test (TUG). RESULTS: Excellent intra-rater, inter-rater and test-retest reliability were found, with a minimal detectable change of 3.26 s. AST times were significantly associated with FMA-LE assessment, FTSTS times, LOS in the forward and backward directions and to the affected side, BBS ratings and TUG times. CONCLUSION: AST time is a reliable assessment tool that correlates with different stroke-specific impairments in people with chronic stroke.
Assuntos
Avaliação da Deficiência , Reabilitação do Acidente Vascular Cerebral , Idoso , Doença Crônica , Estudos Transversais , Feminino , Humanos , Extremidade Inferior/fisiopatologia , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Modalidades de Fisioterapia , Equilíbrio Postural/fisiologia , Centros de Reabilitação , Reprodutibilidade dos Testes , Acidente Vascular Cerebral/fisiopatologiaRESUMO
BACKGROUND: T-box expressed in T cells (TBET) and guanine adenine thymine adenine sequence-binding protein 3 (GATA3) play important roles in the differentiation of Th1 and Th2 subsets, which contributes to the progression of acute coronary syndrome (ACS). OBJECTIVE: This study aimed to investigate the temporal change of TBET/GATA3 mRNA ratio in ACS. METHODS: Thirty-three patients suspected of ACS with symptom onset within 24 hours were recruited. Blood samples were taken after arrival at the emergency department and at hourly intervals until the 6th hour. The mRNA expressions of TBET and GATA3 were quantified by a real-time RT-qPCR. RESULTS: The TBET/GATA3 mRNA ratio was elevated dramatically in patients with acute myocardial infarction (AMI) and exhibited biphasic M-shaped release kinetics with two distinct peaks. The ratio was elevated 2 hours after symptom onset, dropped to the lowest level at 10 hours, and rose to the second peak at 14 hours. A similar biphasic M-shaped curve was observed in AMI patients with blood samples taken prior to any intervention. CONCLUSIONS: The TBET/GATA3 mRNA ratio was elevated in AMI patients throughout most of the first 20 hours after symptom onset. The biphasic M-shaped release kinetics was more likely to reflect pathophysiological changes rather than treatment effects.