Anders Olof Herman Nygren

Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting

OBJECTIVE We sought to evaluate a multiplexed massively parallel shotgun sequencing assay for noninvasive trisomy 21 detection using circulating cell-free fetal DNA. STUDY DESIGN Sample multiplexing and cost-optimized reagents were evaluated as improvements to a noninvasive fetal trisomy 21 detection assay. A total of 480 plasma samples from high-risk pregnant women were employed. RESULTS In all, 480 prospectively collected samples were obtained from our third-party storage site; 13 of these were removed due to insufficient quantity or quality. Eighteen samples failed prespecified assay quality control parameters. In all, 449 samples remained: 39 trisomy 21 samples were correctly classified; 1 sample was misclassified as trisomy 21. The overall classification showed 100% sensitivity (95% confidence interval, 89-100%) and 99.7% specificity (95% confidence interval, 98.5-99.9%). CONCLUSION Extending the scope of previous reports, this study demonstrates that plasma DNA sequencing is a viable method for noninvasive detection of fetal trisomy 21 and warrants clinical validation in a larger multicenter study.

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

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. METHODS The assay used methylation-sensitive restriction enzymes to eliminate the maternal (unmethylated) fraction of the DNA sample. The undigested fetal DNA fraction was then coamplified in the presence of a synthetic oligonucleotide to permit competitive PCR. The amplification products were quantified by single-base extension and MALDI-TOF MS analysis. RESULTS Using 2 independent markers, (sex determining region Y)-box 14 (SOX14) and T-box 3 (TBX3), we measured a mean of 151 copies of fetal DNA/mL plasma and a mean fetal fraction of 0.13 in samples obtained from pregnant women. We investigated 242 DNA samples isolated from plasma from pregnant and nonpregnant women and observed an analytical sensitivity and specificity for the assay of 99% and 100%, respectively. CONCLUSIONS By investigating several regions in parallel, we reduced the measurement variance and enabled quantification of circulating cell-free DNA. Our results indicate that this multiplex methylation-based reaction detects and quantifies the amount of fetal DNA in a sample isolated from maternal plasma.