Predicting gestational diabetes mellitus risk at 11-13 weeks' gestation: the role of extrachromosomal circular DNA.

BACKGROUND: Gestational diabetes mellitus (GDM) significantly impacts maternal and infant health both immediately and over the long term, yet effective early diagnostic biomarkers are currently lacking. Thus, it is essential to identify early diagnostic biomarkers for GDM risk screening. Extrachromosomal circular DNA (eccDNA), being more stable than linear DNA and involved in disease pathologies, is a viable biomarker candidate for diverse conditions. In this study, eccDNA biomarkers identified for early diagnosis and assessment of GDM risk were explored. METHODS: Using Circle-seq, we identified plasma eccDNA profiles in five pregnant women who later developed GDM and five matched healthy controls at 11-13 weeks of gestation. These profiles were subsequently analyzed through bioinformatics and validated through outward PCR combined with Sanger sequencing. Furthermore, candidate eccDNA was validated by quantitative PCR (qPCR) in a larger cohort of 70 women who developed GDM and 70 normal glucose-tolerant (NGT) subjects. A ROC curve assessed the eccDNA's diagnostic potential for GDM. RESULTS: 2217 eccDNAs were differentially detected between future GDM patients and controls, with 1289 increased and 928 decreased in abundance. KEGG analysis linked eccDNA genes mainly to GDM-related pathways such as Rap1, MAPK, and PI3K-Akt, and Insulin resistance, among others. Validation confirmed a significant decrease in eccDNA PRDM16circle in the plasma of 70 women who developed GDM compared to 70 NGT women, consistent with the eccDNA-seq results. PRDM16circle showed significant diagnostic value in 11-13 weeks of gestation (AUC = 0.941, p < 0.001). CONCLUSIONS: Our study first demonstrats that eccDNAs are aberrantly produced in women who develop GDM, including PRDM16circle, which can predict GDM at an early stage of pregnancy, indicating its potential as a biomarker. TRIAL REGISTRATION: ChiCTR2300075971, http://www.chictr.org.cn . Registered 20 September 2023.

Differential expression and analysis of extrachromosomal circular DNAs as serum biomarkers in pulmonary arterial hypertension.

BACKGROUND: Extrachromosomal circular DNAs (eccDNAs) have been reported to play a key role in the occurrence and development of various diseases. However, the characterization and role of eccDNAs in pulmonary arterial hypertension (PAH) remain unclear. METHODS: In the discovery cohort, we first explored eccDNA expression profiles by Circle-sequencing analysis. The candidate eccDNAs were validated by routine polymerase chain reaction (PCR), TOPO-TA cloning and Sanger sequencing. In the validation cohort, 30 patients with PAH and 10 healthy controls were recruited for qPCR amplification to detect the candidate eccDNAs. Datas at the baseline were collected, including clinical background, biochemical variables, echocardiography and hemodynamic factors. Receiver operating characteristic curve was used to investigate the diagnostic effect of the eccDNA. RESULTS: We identified a total of 21,741 eccDNAs in plasma samples of 3 IPAH patients and 3 individuals in good health, and the expression frequency, GC content, length distribution, and genome distribution of the eccDNAs were thoroughly characterized and analyzed. In the validation cohort, 687 eccDNAs were differentially expressed in patients with IPAH compared with healthy controls (screening threshold: |FC|≥2 and P < 0.05). Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the specific eccDNAs in IPAH were significantly enriched in calcium channel activity, the mitogen-activated protein kinase pathway, and the wnt signaling pathway. Verification queue found that the expression of eccDNA-chr2:131208878-131,424,362 in PAH was considerably higher than that in healthy controls and exhibited a high level of accuracy in predicting PAH with a sensitivity of 86.67% and a specificity of 90%. Furthermore, correlation analysis disclosed a significant association between serum eccDNA-chr2:131208878-131,424,362 and mean pulmonary artery pressure (mPAP) (r = 0.396, P = 0.03), 6 min walking distance (6MWD) (r = -0.399, P = 0.029), N-terminal pro-B-type natriuretic peptide (NT-proBNP) (r = 0.685, P < 0.001) and cardiac index (CI) (r = - 0.419, P = 0.021). CONCLUSIONS: This is the first study to identify and characterize eccDNAs in patients with PAH. We revealed that serum eccDNA-chr2:131208878-131,424,362 is significantly overexpressed and can be used in the diagnosis of PAH, indicating its potential as a novel non-invasive biomarker.

Characterization of extrachromosomal circular DNAs in plasma of patients with clear cell renal cell carcinoma.

BACKGROUND AND PURPOSE: Extrachromosomal circular DNAs (eccDNAs) have been recognized for their significant involvement in numerous biological processes. Nonetheless, the existence and molecular characteristics of eccDNA in the peripheral blood of patients diagnosed with clear cell renal cell carcinoma (ccRCC) have not yet been reported. Our aim was to identify potentially marked plasma eccDNAs in ccRCC patients. METHODS AND MATERIALS: The detection of plasma eccDNA in ccRCC patients and healthy controls was performed using the Tn5-tagmentation and next-generation sequencing (NGS) method. Comparisons were made between ccRCC patients and healthy controls regarding the distribution of length, gene annotation, pattern of junctional nucleotide motif, and expression pattern of plasma eccDNA. RESULTS: We found 8,568 and 8,150 plasma eccDNAs in ccRCC patients and healthy controls, respectively. There were no statistical differences in the length distribution, gene annotation, and motif signature of plasma eccDNAs between the two groups. A total of 701 differentially expressed plasma eccDNAs were identified, and 25 plasma eccDNAs with potential diagnostic value for ccRCC have been successfully screened. These up-regulated plasma eccDNAs also be indicated to originate from the genomic region of the tumor-associated genes. CONCLUSION: This work demonstrates the characterization of plasma eccDNAs in ccRCC and suggests that the up-regulated plasma eccDNAs could be considered as a promising non-invasive biomarker in ccRCC.

Categorizing Extrachromosomal Circular DNA as Biomarkers in Serum of Cancer.

Extrachromosomal circular DNA (eccDNA), a double-stranded circular DNA molecule found in multiple organisms, has garnered an increasing amount of attention in recent years due to its close association with the initiation, malignant progression, and heterogeneous evolution of cancer. The presence of eccDNA in serum assists in non-invasive tumor diagnosis as a biomarker that can be assessed via liquid biopsies. Furthermore, the specific expression patterns of eccDNA provide new insights into personalized cancer therapy. EccDNA plays a pivotal role in tumorigenesis, development, diagnosis, and treatment. In this review, we comprehensively outline the research trajectory of eccDNA, discuss its role as a diagnostic and prognostic biomarker, and elucidate its regulatory mechanisms in cancer. In particular, we emphasize the potential application value of eccDNA in cancer diagnosis and treatment and anticipate the development of novel tumor diagnosis strategies based on serum eccDNA in the future.

One-Step Reverse Transcriptase qPCR Method for Serum Hepatitis B Virus RNA Quantification.

In recent years, serum hepatitis B virus (HBV) RNA has been identified as a promising noninvasive surrogate biomarker of intrahepatic covalently closed circular DNA (cccDNA), detection of which requires an invasive liver biopsy in patients with chronic HBV infection. It is impractical to detect intrahepatic cccDNA as a routine diagnosis for chronic hepatitis B (CHB) patients in clinical management. Here, we describe a detailed protocol for serum HBV RNA quantification, which can reflect the activity of intrahepatic cccDNA. The procedure includes three major steps: (1) Simultaneous isolation of HBV DNA and RNA from patients' serum, (2) DNase I digestion for removing HBV DNA contamination, and (3) HBV RNA quantification by one-step reverse transcription qPCR.

Marcadores pronósticos y función inmune en niños VIH - 1 tratados con terapia antirretroviral potente / Prognostic markers and immune function in HIV-1-infected children receiving highly active antiretroviral therapy

La terapia antirretroviral potente (TAP) ha alterado espectacularmente el curso de la infección por el virus de la inmunodeficiencia humana al reducir la replicación viral por debajo de los límites de detección de los ensayos ultrasensibles actuales y provocar un aumento del número de células T CD4+ así como una mejoría clínica. Estos dos marcadores son los más ampliamente utilizados en la monitorización de la eficacia terapéutica en el paciente VIH+. Las respuestas virológicas e inmunológicas a la TAP no son siempre concordantes; lo que sugiere que el grado de recuperación inmunológica tras TAP depende, no sólo del grado de supresión de la replicación viral, sino también de las propiedades citopáticas y de la fitness (capacidad replicatioa) de la cepa viral, así como de la condición subyacente del sistema inmune. Tras la TAP se observan, en muchos casos, aumentos sostenidos en el recuento de CD4+, a pesar de una alta incidencia de 'fallo virológico'. Estos cambios cuantitativos están acompañados por cambios cualitativos en la respuesta inmune y disminución del riesgo de infecciones oportunistas. Se han propuesto varios marcadores de reconstitución inmune. Con la TAP se consigue un incremento de linfocitos T CD4+ y CD8+ de memoria y virgen y disminución de los linfocitos T activados (CD38+, CD25+, HLA-DR+). Esta variación en las subpoblaciones linfocitarias se ve reflejada por un aumento en la generación de fragmentos de ADN circular extracromosómicos (TREC) y en la estabilización del repertorio de TCR (T--cell antigen receptor), que están alterados en la historia natural de la infección. Además de los cambios cuantitativos, también hay cambios cualitativos, como el aumento de la respuesta a antígenos de recuerdo (función de las células de memoria), neoantígenos (función de las células virgen) y antígenos del VIH (respuesta VIH específica) in vivo e in vitro. La supresión de la replicación viral también origina una disminución del número de linfocitos T citotóxicos (CTL) frente al VIH-1 por falta de estímulo antigénico. También hay una caída de la apoptosis celular. Presentamos una revisión actualizada de los marcadores implica-dos en la reconstitución inmunológica en pacientes con VIH (AU)