Maternal Constitutional and Acquired Copy Number Variations

Introduction

Extracellular nucleic acids have already been known to exist in the blood circulation since 1948 . The observation of abnormally high concentrations of DNA in serum of cancer patients lead Stroun et al. to suggest that DNA found in the serum of cancer patients might be released by tumor cells . This hypothesis was confirmed when tumor-derived oncogene mutations were identified in the plasma and serum of patients with hematological malignancies . Since then, analysis of cell-free DNA in plasma identified copy number alterations, single nucleotide variants, and viruses associated with several types of cancers . Similarly, the placenta is considered by many a pseudomalignant tissue, invading the uterine epithelium to enable embryo implantation while releasing fragments of DNA in the maternal plasma as part of a physiological placental cell turnover during pregnancy . Inspired by this theory, Lo et al. discovered Y-chromosomal sequences in the plasma of pregnant women with male fetuses in 1997 . In healthy pregnant women, the majority of plasma DNA is a result of apoptosis or necrosis of maternal cells from multiple tissues . In contrast, “fetal” DNA constitutes only a minor fraction of approximately 5%–20% of cfDNA in plasma during the first trimester, and originates mainly from the cytotrophoblast cells of the placenta . Therefore any noninvasive prenatal test contains a major signal from the maternally derived DNA and a minor signal from the placenta-derived DNA. When applying genome-wide sequencing of plasma cfDNA not only the placental genome and potential aneuploidies in the conceptus are identified, but also the maternal genome is scanned for differences compared to the normal.

Early reports on the size of cfDNA using qPCR have shown that placenta-derived DNA fragments were generally shorter than the maternal ones . More recently, paired-end sequencing has allowed to determine the exact size of millions of cell-free DNA fragments in plasma and distinguished unique placental reads from the high background of maternal DNA. The predominant size of plasma DNA of pregnant women is 166 bp followed by a series of smaller peaks that present at a periodicity of 10 bp at sizes of 143 bp and shorter . It has been shown that the shorter placental DNA fragments are overrepresented in the fraction with fragments smaller than 150 bp . It has also been shown that DNA degradation is not random but occurs by enzymatic processes at specific sites between nucleosomes, the internucleosomal linker sites. These patterns of cfDNA release have been studied extensively by two groups. Snyder et al. demonstrated that nucleosome footprints in cfDNA are tissue specific. They inferred nucleosome spacing from cfDNA in healthy individuals and demonstrated that it correlates with epigenetic features of lymphoid and myeloid cells. This finding reinforces the idea that hematopoietic cell death is the main source of cfDNA . The group of Lo performed plasma DNA tissue mapping, a technique based on genome-wide bisulfite sequencing of plasma DNA. This analysis revealed the major tissue contributors of circulating DNA in both healthy nonpregnant and pregnant women as well as in patients with cancer. Similarly to Snyder et al. they found that > 70% of the circulating DNA is derived from white blood cells, neutrophils, and lymphocytes. The remaining fraction is mainly derived from the liver and, in pregnant women, the placenta. The authors speculate that with further refinements in the deconvolution algorithm, it might become possible to identify the contribution of other tissues to the DNA pool . Hence, localization of the cfDNA fragments can reveal the tissue of origin that contributes to cfDNA in both healthy and pathological conditions.

Maternal Constitutional Copy Number Variations

Because maternal DNA constitutes the major DNA fraction of cfDNA, it may not be surprising that discrepancies of the maternal genome with respect to the reference genome can interfere with the interpretation of cfDNA-based prenatal testing results. Maternal constitutional copy number variations (CNVs) have been a major contribution to false positive cfDNA-based prenatal test results . Our group was the first to show that CNVs as small as 500 kb can cause trisomy z -scores to pass the accepted cutoff of three leading to false positive or false negative results . The group of Snyder demonstrated that a trisomy 18 discordancy between noninvasive and invasive prenatal test results was caused by a constitutional large duplication on the maternal chromosome 18 and, in another case, by a smaller maternal CNV of approximately 500 kb . In a recent systematic review of the literature it was compiled that among the 60 false positive trisomy cases, 29 (48%) had an underlying maternal CNV . Zhou et al. showed that there is a strong correlation of higher z-scores with increasing size of maternal CNVs . Similarly, maternal X chromosomal CNVs have been reported to be the cause of false positive sex chromosome aneuploidy cfDNA-based prenatal test results .

Because placental DNA constitutes only a small fraction of plasma cfDNA, not only constitutional (i.e., present in all cells of the body), but also somatic CNVs (i.e., present in only a subset of all cells) can cause false positive and, theoretically, false negative results. Maternal (segmental) chromosomal mosaicism is another major cause of discordant results of cfDNA screening and even low-grade mosaicism in the maternal genome can skew the z -statistics significantly. Bayindir et al. identified a maternal mosaic segmental deletion on chromosome 13 by applying genome-wide analysis. This deletion was present in only 17% of the maternal blood cells analyzed by FISH . Although rare, similar large mosaic maternal duplications of the chromosome 18 long arm have been observed and they caused false positive cfDNA test results . In another case with a false positive cfDNA test for fetal trisomy 18, the mother was identified with a supernumerary ring chromosome 18 in 35% of her cells .

Cell-free DNA-based prenatal testing in pregnancies with sex chromosome aneuploidies (SCAs) has lower sensitivities and specificities as compared to testing for the common fetal trisomies . In a prospective study, Wang et al. have shown that maternal chromosome X mosaicism was the main cause of discordant sex chromosomal aneuploidy . In another study by the same group, cfDNA Noninvasive Prenatal Testing (NIPT) findings suggested a case with lower X chromosome concentration. Maternal karyotyping showed mosaicism for monosomy X (mosaic Turner syndrome), indicating that this maternal mosaicism masked the true contribution of fetal chromosome X to the cell-free DNA pool .

The relatively high frequency of maternal CNVs and maternal sex chromosome mosaicism warrants maternal white blood cell testing with array-CGH or FISH to avoid unnecessary invasive testing . Most maternal CNVs are benign or of unknown clinical significance .