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. INTRODUCTIONThe presence of circulating cell-free fetal DNA in the maternal plasma of the pregnant woman, 1 in combination with recent advances in massively parallel sequencing (MPS) technologies, has made noninvasive prenatal testing (NIPT) of fetal aneuploidy a reality. NIPT reduces the need for invasive sampling and the associated risk of procedure-related pregnancy loss. In 2008, it was demonstrated that noninvasive fetal aneuploidy detection by MPS was feasible. 2,3 Multiple clinical validation studies using either targeted or whole-genome sequencing demonstrated the high sensitivity and specificity of NIPT. [4][5][6][7][8][9][10][11][12][13][14][15] Although most validation studies were predominantly evaluating the clinical validity in pregnancies at increased risk of the most common aneuploidies, it was recently shown that screening all pregnant women has positive predictive values of 45.5% and 40% for detection of trisomies 21 and 18, respectively. 16 MPS for aneuploidy detection applies counting statistics to millions of sequencing reads to identify subtle changes in the small percentage of fetal DNA present in the total cell-free DNA isolated from maternal plasma. 17,18 An increase or decrease in the number of normalized sequencing reads, typically converted to a 'z-score', 18 a 'normalized chromosome value', 13 genome-wide normalized score 19 or by 'withinsample copy number aberration detector' 20 is indicative of aneuploidy for the respective chromosome. Despite the high accuracy of current NIPT testing, a baseline false-positive and false-negative rate remains. Those incorrect results may have both biological and technical causes:

Contribution of maternal copy number variations to false‐positive fetal trisomies detected by noninvasive prenatal testing

“…Follow‐up case studies of false‐positive NIPT results has identified a variety of potential causes including a vanishing twin, unidentified maternal tumors, maternal mosaicism, and confined placental mosaicism . More recently, several groups using different NIPT methodologies have reported maternal copy number variations (CNVs) as an additional cause of false‐positive NIPT results . In order to further investigate the potential contribution of maternal CNVs to false‐positive NIPT results, we performed NIPT on a large cohort of 112 021 pregnant women using low coverage massively parallel sequencing and analyzed the 74 false‐positive trisomy samples for the presence of maternal CNVs.…”

Cytogenetic confirmation of a positive NIPT result: evidence-based choice between chorionic villus sampling and amniocentesis depending on chromosome aberration

“…Moreover, there are already reports on genome-wide NIPT analysis with promising results [2,3]. For trisomy 13,18, and 21 screening, the test performs much better than the traditional first trimester screening (combined test) [1].…”

“…However, in order to avoid the abortion of an unaffected fetus it then will be necessary not only to rely on z-scoring, but to use an analysis pipeline that allows whole chromosome analysis. This enables differentiation of high z-scores due to fetal trisomy from those caused by maternal CNVs [2,46,47]. Moreover, also other causes of false positive NIPT results, such as maternal mosaicism or a vanishing twin, should be excluded in such cases before TOP may be considered.…”

Presymptomatic Identification of Cancers in Pregnant Women During Noninvasive Prenatal Testing

“…[4][5][6] We optimized a large parallel sequencing-based NIPT dataset and analysis, which not only interrogates the common trisomies but also allows the genomewide discrimination of fetal and maternal segmental aneuploidies. 3 All patients undergoing NIPT consented to release of information for study purposes beyond trisomy 13, 18, and 21; this consent and the study protocol were approved by the University Hospitals, Leuven ethical board. Of the first 4000 prospective NIPT samples, we identified 3 profiles with an aberrant quality score and reproducible genomewide representation (GR) profiles reminiscent of cancer-related copy number variation.…”