Digital PCR Assay Utilizing In-Droplet Methylation-Sensitive Digestion for Estimation of Fetal cfDNA From Plasma.

IF 2.7 2区医学 Q2 GENETICS & HEREDITY

Prenatal Diagnosis Pub Date : 2025-03-16 DOI:10.1002/pd.6774

Richard Dannebaum, Olga Mikhaylichenko, David Siegel, Chenyu Li, Eric Hall, Severine Margeridon, Monica Herrera, Kristin Loomis, Thea Riel, Madhumita Ramesh, Maria Gencoglu, Nathan Hendel, Anthony Henriquez, Nyari Dzvova, Raymond-John Abayan, Xinhua Lin, Martin Chavez, Nazeeh Hanna

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引用次数: 0

Abstract

Objective: Recent guidelines suggest that non-invasive prenatal screening (NIPS) should be offered to all patients with singleton and twin pregnancies. Accurate determination of fetal fraction in cell-free DNA (cfDNA) is vital for reliable NIPS outcomes. We propose a methylation-based approach using droplet digital PCR (ddPCR) and methylation-sensitive restriction enzyme (MSRE) digestion for fetal fraction quantification as an affordable and fast solution.

Method: Following biomarker discovery using early pregnancy placental genomic DNA (gDNA) and cfDNA from non-pregnant female individuals, we designed assays targeting MSRE-compatible regions based on contrasting methylation patterns between maternal and fetal cfDNA. We established a proof-of-concept ddPCR workflow on the Bio-Rad Droplet Digital PCR QX600 instrument.

Results: Testing the fetal fraction assay multiplex on 137 prospective clinical samples demonstrated high concordance with NGS results for both female and male pregnancies as well as with chromosome Y-based calculations for samples with a male fetus. Reproducibility analysis indicated lower variability compared to previously reported NGS performance.

Conclusion: This study showcases the potential of this novel, 6-color, high-multiplex methylation ddPCR panel for accurate measurement of fetal fraction in cfDNA samples. It presents opportunities to integrate such methodology as a standalone measurement to assess the quality of samples undergoing NIPS.

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来源期刊

Prenatal Diagnosis 医学-妇产科学

CiteScore

5.80

自引率

13.30%

发文量

204

审稿时长

2 months

期刊介绍： Prenatal Diagnosis welcomes submissions in all aspects of prenatal diagnosis with a particular focus on areas in which molecular biology and genetics interface with prenatal care and therapy, encompassing: all aspects of fetal imaging, including sonography and magnetic resonance imaging; prenatal cytogenetics, including molecular studies and array CGH; prenatal screening studies; fetal cells and cell-free nucleic acids in maternal blood and other fluids; preimplantation genetic diagnosis (PGD); prenatal diagnosis of single gene disorders, including metabolic disorders; fetal therapy; fetal and placental development and pathology; development and evaluation of laboratory services for prenatal diagnosis; psychosocial, legal, ethical and economic aspects of prenatal diagnosis; prenatal genetic counseling