Sequencing of Circulating Cell-free DNA during Pregnancy

Abstract

Sequence analysis of cell-free DNA (cfDNA) fragments that circulate in the blood of pregnant women, along with the translation of this method into screening for fetal chromosome abnormalities, is a success story of modern genomic medicine. In less than a decade, prenatal cfDNA testing has gone from small, proof-of-principle studies to a global transformation of prenatal care. As of late 2017, a total of 4 million to 6 million pregnant women had had DNA from their plasma analyzed to screen for fetal aneuploidy. The exponential growth of the test has been a function of the role of the biotechnology industry in its development and marketing. Here we review what has been learned from the wide-scale implementation of this testing, how it has changed prenatal clinical care, and what ethical concerns have arisen, and we speculate about what lies ahead.

Figure 1.. The Two Main Methods of Maternal Plasma DNA Sequencing for Prenatal Screening of Fetal Chromosomal Aneuploidies.

Testing for trisomy 21 is shown as an illustrative example. In random sequencing, cell-free DNA (cfDNA) fragments originating from any chromosome are sequenced. A random representative selection of maternal (purple fragments) and fetal (orange fragments) cfDNA molecules is sequenced. The DNA molecules belonging to different human chromosomes are counted to determine the proportion of cfDNA molecules derived from chromosome 21. The proportion of chromosome 21 DNA sequences is elevated if the plasma sample was collected from a pregnant woman carrying a fetus with trisomy 21. In targeted sequencing, only cfDNA from specific chromosomes of interest is sequenced. Loci of single-nucleotide polymorphisms (SNPs, highlighted in blue) on targeted chromosomes are amplified and sequenced. Fetal cfDNA from chromosome 21 is indicated by orange fragments with blue highlighting. Maternal cfDNA from chromosome 21 is indicated by purple fragments with blue highlighting. Ratios between heterozygous SNP alleles on the cfDNA from chromosome 21 are compared with ratios similarly computed for other targeted chromosomes. If the fetus is aneuploid, allelic ratios on the aneuploid chromosome are skewed in comparison with other, nonaneuploid chromosomes.

Figure 2.. General Methodologic Principles of Noninvasive Prenatal Tests for Fetal Single-Gene Diseases.

A man with alleles 1 and 2 and a woman with alleles 3 and 4 for an autosomal recessive disease locus seek noninvasive prenatal diagnosis. Maternal plasma is sampled, and cfDNA fragments originating from the disease locus are examined. Some of the cfDNA molecules originate from the fetus and are inherited from the father. Blue fragments corresponding to DNA bearing the paternal allele 2 are detected. This suggests that the fetus has inherited allele 2 from the father. The majority of the remaining DNA fragments are derived from the mother. These DNA molecules could bear either maternal allele 3 (pink fragments) or allele 4 (purple fragments). The amount of cfDNA bearing allele 3 should be almost equal to the amount of cfDNA bearing allele 4 if not for the presence of fetal DNA. In this example, there are more purple fragments in the sample than pink fragments. This suggests that the fetus has inherited allele 4 from the mother. The quantitative comparison between maternal alleles 3 and 4 could be based on the relative ratio between alleles at a single polymorphic or mutated locus or among alleles at multiple loci belonging to either haplotype.