학술논문
Precision of Fetal DNA Fraction Estimation by Quantitative Polymerase Chain Reaction Quantification of a Differently Methylated Target in Noninvasive PrenatalTesting
Science
Science
Document Type
Academic Journal
Source
Laboratory Medicine. May 2020, Vol. 51 Issue 3, p279, 9 p.
Subject
Language
English
ISSN
0007-5027
Abstract
Plasma circulating cell-free DNA (ccfDNA) from pregnant women contains fragments of fetal origin, specifically from placental trophoblastic cells. (1) The proportion of the total ccfDNA of a specimen of fetal [...]
Background: The performance of noninvasive prenatal testing (NIPT) assays is critically determined by the proportion of fetal DNA or fetal fraction (FF). Fetomaternal differential methylation of certain genomic regions has been proposed as a universal marker of fetal origin, and previous reports have suggested the use of methylation-sensitive restriction enzyme (MSRE) assays to estimate FF. Methods: We analyzed the performance of FF estimation using an MSRE assay with duplex quantitative polymerase chain reaction (qPCR). Mixtures of genomic DNA from placental cells and from adult women were digested with 2 MSRE and FF estimates obtained, for a total of 221 pairwise treatment/control comparisons. Results: The coefficient of variance (CV) of the MSRE assays was high, ranging from 24% to 60%. An alternative in silico FF estimation algorithm, SeqFF, displayed slightly lower variability, with a CV of 22%. Conclusion: These results cast doubts on the usefulness of the MSRE-based assay of differentially methylated markers for FF estimation. The lack of a universal method capable of precisely estimating FF remains an incompletely solved issue. Keywords: fetal DNA, ccffDNA, fetal fraction, NIPT, methylation, MSRE, noninvasive prenatal testing, Down syndrome, next-generation sequencing
Background: The performance of noninvasive prenatal testing (NIPT) assays is critically determined by the proportion of fetal DNA or fetal fraction (FF). Fetomaternal differential methylation of certain genomic regions has been proposed as a universal marker of fetal origin, and previous reports have suggested the use of methylation-sensitive restriction enzyme (MSRE) assays to estimate FF. Methods: We analyzed the performance of FF estimation using an MSRE assay with duplex quantitative polymerase chain reaction (qPCR). Mixtures of genomic DNA from placental cells and from adult women were digested with 2 MSRE and FF estimates obtained, for a total of 221 pairwise treatment/control comparisons. Results: The coefficient of variance (CV) of the MSRE assays was high, ranging from 24% to 60%. An alternative in silico FF estimation algorithm, SeqFF, displayed slightly lower variability, with a CV of 22%. Conclusion: These results cast doubts on the usefulness of the MSRE-based assay of differentially methylated markers for FF estimation. The lack of a universal method capable of precisely estimating FF remains an incompletely solved issue. Keywords: fetal DNA, ccffDNA, fetal fraction, NIPT, methylation, MSRE, noninvasive prenatal testing, Down syndrome, next-generation sequencing