DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study

Abstract

Purpose: To determine whether maternal plasma cell-free DNA sequencing can effectively identify trisomy 18 and 13.

Methods: Sixty-two pregnancies with trisomy 18 and 12 with trisomy 13 were selected from a cohort of 4,664 pregnancies along with matched euploid controls (including 212 additional Down syndrome and matched controls already reported), and their samples tested using a laboratory-developed, next-generation sequencing test. Interpretation of the results for chromosome 18 and 13 included adjustment for CG content bias.

Results: Among the 99.1% of samples interpreted (1,971/1,988), observed trisomy 18 and 13 detection rates were 100% (59/59) and 91.7% (11/12) at false-positive rates of 0.28% and 0.97%, respectively. Among the 17 samples without an interpretation, three were trisomy 18. If z-score cutoffs for trisomy 18 and 13 were raised slightly, the overall false-positive rates for the three aneuploidies could be as low as 0.1% (2/1,688) at an overall detection rate of 98.9% (280/283) for common aneuploidies. An independent academic laboratory confirmed performance in a subset.

Conclusion: Among high-risk pregnancies, sequencing circulating cell-free DNA detects nearly all cases of Down syndrome, trisomy 18, and trisomy 13, at a low false-positive rate. This can potentially reduce invasive diagnostic procedures and related fetal losses by 95%. Evidence supports clinical testing for these aneuploidies.

Figure 1

Flow chart showing the cohort of samples collected, those selected for testing, and the numbers for which testing was successful. This report focuses on 1,988 pregnancies subject to testing. Of these, 286 were common trisomies (Down syndrome, trisomy 18, and trisomy 13), along with 1,702 matched euploid controls.

Figure 2

z-Scores for chromosomes 18, 13, and 21. (a) Chromosome 18 z-scores for 59 pregnancies with trisomy 18 and for pregnancies without trisomy 18 versus fetal fraction. Large squares indicate a trisomy 18 pregnancy. Small circles, diamonds, and triangles indicate euploid, Down syndrome, and trisomy 13 pregnancies, respectively. One z-score above 40 was truncated to 39.9 for display. (b) Chromosome 13 z-scores for 12 pregnancies with trisomy 13 and for pregnancies without trisomy 13 versus fetal fraction. Large triangles indicate a trisomy 13 pregnancy. Small circles, diamonds, and squares indicate euploid, Down syndrome, and trisomy 18 pregnancies, respectively. One z-score above 40 was truncated to 39.9 for display. Note the false-negative result at a z-score of 0 and fetal fraction of 13%. (c) Chromosome 21 interpretations z-scores for 212 Down syndrome pregnancies and for pregnancies without Down syndrome versus fetal fraction. Large diamonds indicate a Down syndrome pregnancy. Small circles, squares, and triangles indicate euploid, trisomy 18, and trisomy 13 pregnancies, respectively.

Figure 3

Interpreting chromosome 18, 13, and 21 sequencing results as z-scores versus multiples of the median (MoM): comparing and exploring appropriate cutoff levels. This figure shows the chromosome-specific z-score results are on the x-axis, with the same data expressed as multiples of the plate-specific median level on the y-axis. Results for euploid, trisomy 18, trisomy 13, and Down syndrome pregnancies are shown as small circles, squares, triangles, and diamonds, respectively. The aneuploidy of interest (e.g., trisomy18 for chromosome 18 results) is shown with a larger symbol size. The top row of (a) and (b) shows the chromosome 18 results, while (c) and (d) and (e) and (f) show results for chromosome 13 and 21, respectively. The figures on the right hand side show the same data but focus on the results near the cutoff levels. The vertical dashed line shows the original cutoff levels chosen to validate the laboratory-developed test. The gray regions show where cutoff levels might provide better screening performance in the future.