Noninvasive Detection of Fetal Trisomy 21 by Sequencing of DNA in Maternal Blood: A Study in a Clinical Setting

The incidence of trisomy 21 in the general population is about 1 in 800 births. The risk of this chromosomal aneuploidy increases with age to 1 in 35 term births for women 45 years of age. The risk can increase substantially when advanced maternal age is integrated with other maternal risk factors, including positive serum screening results, fetal ultrasound abnormality, or family history. Currently utilized tests to confirm the diagnosis of trisomy 21 in high-risk women are invasive procedures, including genetic amniocentesis and chorionic villus sampling. Although the safety of these invasive procedures has greatly improved, there is still risk of iatrogenic fetal loss, albeit low. Noninvasive methods to diagnose fetal trisomy 21 are being actively investigated to identify plasma samples with an overrepresentation of DNA from chromosome 21. One such method is multiplexed massively parallel shotgun sequencing (MPSS), which has been demonstrated to successfully identify women carrying fetuses with trisomy 21 by analyzing circulating cell-free fetal DNA in maternal plasma. Preliminary data using this assay are promising and have shown that MPSS can distinguish women carrying a trisomy 21 fetus from those carrying a euploid fetus. However, only small numbers of plasma samples have been studied to date, and thus far the costs associated with the assay have been prohibitive with respect to potential deployment in clinical practice.This blinded prospective study evaluated a modified MPSS assay for noninvasive trisomy 21 detection, analyzing circulating cell-free fetal DNA in plasma samples from pregnant woman at high risk for fetal aneuploidy. Important modifications made to the sequencing protocol included sample multiplexing, use of custom-purified enzymes, and cost-optimized reagents. These modifications achieved a marked reduction in assay cost.A total of 480 plasma samples from pregnant woman at high risk for fetal aneuploidy were prospectively collected, processed, and stored at a third-party location. Of the 480 samples, 13 were excluded because of the insufficient sample volume or poor quality. Eighteen additional samples were excluded because of failure to meet prespecified quality control parameters for the assay. Of the remaining 449 samples, all 39 of the trisomy 21 pregnancies were correctly identified. One sample from a euploid pregnancy was misclassified as trisomy 21 (false positive); therefore, the test achieved a sensitivity of 100% (95% confi