Noninvasive prenatal testing using a novel analysis pipeline to screen for all autosomal fetal aneuploidies improves pregnancy management

Abstract

Noninvasive prenatal testing by massive parallel sequencing of maternal plasma DNA has rapidly been adopted as a mainstream method for detection of fetal trisomy 21, 18 and 13. Despite the relative high accuracy of current NIPT testing, a substantial number of false-positive and false-negative test results remain. Here, we present an analysis pipeline, which addresses some of the technical as well as the biologically derived causes of error. Most importantly, it differentiates high z-scores due to fetal trisomies from those due to local maternal CNVs causing false positives. This pipeline was retrospectively validated for trisomy 18 and 21 detection on 296 samples demonstrating a sensitivity and specificity of 100%, and applied prospectively to 1350 pregnant women in the clinical diagnostic setting with a result reported in 99.9% of cases. In addition, values indicative for trisomy were observed two times for chromosome 7 and once each for chromosomes 15 and 16, and once for a segmental trisomy 18. Two of the trisomies were confirmed to be mosaic, one of which contained a uniparental disomy cell line. As placental trisomies pose a risk for low-grade fetal mosaicism as well as uniparental disomy, genome-wide noninvasive aneuploidy detection is improving prenatal management.

Figure 1

An example report of a trisomy 21 sample. Details are shown for chromosomes 13, 18 and 21, and for each chromosomes z, zz and BM scores are indicated. When aneuploidy is present, the OM is shown for comparison with BM score. Dot plots of z- vs zz-score (Y and X axis, respectively) are shown with undetermined zones marked (2.5–3) with grey lines. Z-scores of 5 Mb sliding windows are plotted across the chromosome to visualize the aberrations, dotted lines represent ±1.5 z-scores and the areas above and below these cutoff values are coloured in green and red, respectively. Violin plots of BM and OM with their respective median shown with a single dot represent the distribution of all the bin-level z-scores.

Figure 2

Four examples of maternal CNVs that alter counting statistics causing potential false-positive results. For every sample, the NIPT (top) result and the aCGH (bottom) result is shown: (a) 2.55 Mb deletion on chromosome 22q11.21 (chr21.hg19: g.18894820–21440515del); (b) 1.38 Mb duplication on chromosome 13q33.1–q33.2 (chr13.hg19: g.104397732–1057779818dup); (c) 1.73 Mb duplication on chromosome 13q31.3 (chr13.hg19: g.90201888–91936194dup); (d) 1.03 Mb duplication on chromosome 21q11.2–q21.1 (chr21.hg19: g.15485038–16515907dup). While duplications and deletions affect both z- and zz-score, the BM remains in a normal range. The sliding plots help visualize the localization of the aberration as well as the effect.

Figure 3

Z-score vs zz-score (left), and BM vs zz-score (right) for chromosomes 21 and 18 results from all validation study samples. This highlights how the combined analysis reduces potential false-positive and false-negative cases. Triangles and boxes represent trisomies 18 and 21, respectively. Full diamond shape is the mosaic trisomy 18 case, whereas the hollow diamond shapes represent the undetermined trisomy 18 cases. Hollow circles represent the normal population, and full circles represent the cases classified as undetermined by the automated pipeline. The shaded lines on the X and Y axis mark the inconclusive zones between the cutoff values of 2.5–3 for both z- and zz-score, and 1–1.5 for the BM.

Figure 4

Dot plot of zz-scores (Y axis) vs BM (X axis) for all diagnostic study samples for chromosomes 21, 18, 7, 15 and 16. Blue circles represent the normal samples and black circles represent the cases classified as undetermined from automated pipeline. The red boxes represent the cases classified as trisomy for the respective chromosome.

Figure 5

Results of the partial trisomy 18 case. NIPT result for chromosome 18 (top). aCGH result for chromosome 18 (bottom) and FISH result (right) of amniotic fluid cells.