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Lab Med. 2019 Nov 22. pii: lmz068. doi: 10.1093/labmed/lmz068. [Epub ahead of print]

Precision of Fetal DNA Fraction Estimation by Quantitative Polymerase Chain Reaction Quantification of a Differently Methylated Target in Noninvasive Prenatal Testing.

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Medical Biochemistry Service, Department of Laboratory Medicine, CHU (Centre hospitalier universitaire) de Québec-Université Laval, Quebec City, Quebec, Canada.
Department of Molecular Biology, Medical Biochemistry, and Pathology, Faculty of Medicine, Université Laval, Quebec City, Quebec, Canada.
Human and Molecular Genetics Research Unit, Research Center, CHU de Québec, Quebec City, Quebec, Canada.
PEGASUS (Personalized Genomics for Prenatal Abnormalities Screening Using Maternal Blood), Quebec City, Quebec, Canada.



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.


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.


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%.


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.


fetal DNA; Down syndrome; MSRE; NIPT; ccffDNA; fetal fraction; methylation; next-generation sequencing; noninvasive prenatal testing


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