Copy-number variation and false positive prenatal aneuploidy screening results

Abstract

Investigations of noninvasive prenatal screening for aneuploidy by analysis of circulating cell-free DNA (cfDNA) have shown high sensitivity and specificity in both high-risk and low-risk cohorts. However, the overall low incidence of aneuploidy limits the positive predictive value of these tests. Currently, the causes of false positive results are poorly understood. We investigated four pregnancies with discordant prenatal test results and found in two cases that maternal duplications on chromosome 18 were the likely cause of the discordant results. Modeling based on population-level copy-number variation supports the possibility that some false positive results of noninvasive prenatal screening may be attributable to large maternal copy-number variants. (Funded by the National Institutes of Health and others.).

Figure 1. The Role of Maternal Copy-Number Variants (CNVs) in False Positive Results of DNA-based Noninvasive Prenatal Screening

Panel A is a schematic representation of cell-free DNA (cfDNA) analysis. The cfDNA in maternal plasma contains primarily maternal cfDNA and a smaller proportion of fetal cfDNA. The threshold for triggering a positive cfDNA test is indicated by the vertical dashed line. The combination of fetal diploidy and the absence of a maternal CNV results in a true negative test. The combination of fetal trisomy and the absence of a maternal CNV results in a true positive test. The combination of fetal diploidy and the presence of a maternal CNV that duplicates a portion of a relevant chromosome results in a false positive test. Hypothetically, the combination of fetal trisomy on a specific chromosome and the presence of a maternal CNV that deletes a portion of the same chromosome could result in a false negative test. Panel B is a schematic representation of the effect of a maternal CNV on the probability of a false positive test result, expressed as Pr(Z>4.0), which equals approximately 3 in 100,000. Maternal duplications shift the sampling distribution of the test to the right, and the underlying reference distribution is unchanged. In Panel C, copy-number profiles based on normalized cfDNA read depth are consistent with duplicated regions on chromosome 18 in two of the four patients. Profiles of Patients 2 and 4 are consistent with two copies throughout the region of interest. Patients 1 and 3 have an increased copy number in contiguous regions, suggestive of duplications.

Figure 2. Population Frequency and Estimated Effect of Maternal CNVs on False Positive Test Rates

The burden of nonpathogenic copy-number increases on chromosomes 13 (Panel A), 18 (Panel B), and 21 (Panel C) in a cohort of 19,584 persons, predominantly of European ancestry, is shown for a range of CNV sizes (blue circles, right vertical axis). CNV frequencies in each size bin refer to CNVs of the given size or larger. For each size bin, the estimated factor increase in the probability of a false positive test resulting from the copy-number increase is shown for a range of fetal fractions (gray and colored lines, left vertical axis). The sizes of the CNVs present on chromosome 18 in Patients 1 and 3 are highlighted (dashed vertical lines).