Quantification of Fetal DNA by Use of Methylation-Based DNA Discrimination

Abstract

BACKGROUND:Detection of circulating cell-free fetal nucleic acids in maternal plasma has been used in noninvasive prenatal diagnostics. Most applications rely on the qualitative detection of fetal nucleic acids to determine the genetic makeup of the fetus. This method leads to an analytic dilemma, because test results from samples that do not contain fetal DNA or are contaminated with maternal cellular DNA can be misleading. We developed a multiplex approach to analyze regions that are hypermethylated in placenta relative to maternal blood to evaluate the fetal portion of circulating cell-free DNA isolated from maternal plasma.

Current status of non-invasive prenatal testing (NIPT): genetic counseling, dominant methods and overall performance

“…But it is pivotal to know by which method fetal fraction is measured and how precise the measured value is. To date, there are four concepts to determine fetal fraction: quantitation of Y-chromosomal sequences [17], copy number differences [18], differential methylation [19] and polymorphic SNP allele dosage [20]. Quantifying Y-chromosomal cfDNA is only feasible in pregnancies with a male fetus and constitutively underestimates fetal fraction in comparison to a SNP allele dosage method [21,22].…”

Current Concepts in Noninvasive Prenatal Screening (NIPS)

“…Some methods work for both male and female fetuses, but are indirect approaches which exploit characteristics that vary depending on the origin of the DNA. For example, the average length of the cfDNA fragments in a sample has been used to estimate the fetal fraction (fetal fragments are, on average, shorter than those of maternal origin [22]) as has the presence of methylation (fetal fragments are more likely to be methylated than maternal ones [23]). Using SNPs to distinguish fetal from maternal DNA allows a more direct assessment of the proportion of the DNA that is of placental origin.…”

Quality assurance and standardization in view of non-invasive prenatal testing (NIPT)

“…Thus, it is essential for further analysis to implement strategies to measure the quantity of the fetal DNA [4,22]. For instance, the fetal fraction could be determined based on differential methylation [24].…”