Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma

Abstract

Chromosomal aneuploidy is the major reason why couples opt for prenatal diagnosis. Current methods for definitive diagnosis rely on invasive procedures, such as chorionic villus sampling and amniocentesis, and are associated with a risk of fetal miscarriage. Fetal DNA has been found in maternal plasma but exists as a minor fraction among a high background of maternal DNA. Hence, quantitative perturbations caused by an aneuploid chromosome in the fetal genome to the overall representation of sequences from that chromosome in maternal plasma would be small. Even with highly precise single molecule counting methods such as digital PCR, a large number of DNA molecules and hence maternal plasma volume would need to be analyzed to achieve the necessary analytical precision. Here we reasoned that instead of using approaches that target specific gene loci, the use of a locus-independent method would greatly increase the number of target molecules from the aneuploid chromosome that could be analyzed within the same fixed volume of plasma. Hence, we used massively parallel genomic sequencing to quantify maternal plasma DNA sequences for the noninvasive prenatal detection of fetal trisomy 21. Twenty-eight first and second trimester maternal plasma samples were tested. All 14 trisomy 21 fetuses and 14 euploid fetuses were correctly identified. Massively parallel plasma DNA sequencing represents a new approach that is potentially applicable to all pregnancies for the noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.

Fig. 1.

Schematic illustration of the procedural framework for using massively parallel genomic sequencing for the noninvasive prenatal detection of fetal chromosomal aneuploidy. Fetal DNA (thick red fragments) circulates in maternal plasma as a minor population among a high background of maternal DNA (black fragments). A sample containing a representative profile of DNA molecules in maternal plasma is obtained. In this study, one end of each plasma DNA molecule was sequenced for 36 bp using the Solexa sequencing-by-synthesis approach. The chromosomal origin of each 36-bp sequence was identified through mapping to the human reference genome by bioinformatics analysis. The number of unique (U0–1–0–0, see text) sequences mapped to each chromosome was counted and then expressed as a percentage of all unique sequences generated for the sample, termed % chrN for chromosome N. Z-scores for each chromosome and each test sample were calculated using the formula shown. The z-score of a potentially aneuploid chromosome is expected to be higher for pregnancies with an aneuploid fetus (cases E–H shown in green) than for those with a euploid fetus (cases A–D shown in blue).

Fig. 2.

Bar chart of % U0–1–0–0 sequences per chromosome for a maternal plasma sample involving a female fetus (sample 1), a maternal plasma sample involving a male fetus (sample 2), and a mixture of plasma from two adult males (sample 3) processed using the new (protocol A) and original (protocol B) protocols. The percentage of genomic representation of each chromosome as expected for a repeat-masked reference haploid female genome was plotted as a reference (black bars).

Fig. 3.

Plot of (A) % U0–1–0–0 counts and (B) z-scores for chromosome 21 and chromosome X for 28 maternal plasma samples. The sample numbers correspond to the cases described in Table S5.