Objective
The purpose of this study was to assess the prenatal detection rate of trisomy 21 and 18 and the false-positive rate by chromosome-selective sequencing of maternal plasma cell–free DNA.
Study Design
Nested case-control study of cell-free DNA was examined in plasma that was obtained at 11-13 weeks before chorionic villous sampling from 300 euploid pregnancies, 50 pregnancies with trisomy 21, and 50 pregnancies with trisomy 18. Laboratory personnel were blinded to fetal karyotype.
Results
Risk scores for trisomy 21 and 18 were given for 397 of the 400 samples that were analyzed. In all 50 cases of trisomy 21, the risk score for trisomy 21 was ≥99%, and the risk score for trisomy 18 was ≤0.01%. In all 50 cases of trisomy 18, the risk score for trisomy 21 was ≤0.01%, and the risk score for trisomy 18 was ≥99% in 47 cases, 98.8% in 1 case, 88.5% in 1 case, and 0.11% in 1 case. In 3 of the 300 euploid pregnancies (1%), no risk score was provided, because there was failed amplification and sequencing. In the remaining 297 cases, the risk score for trisomy 21 was ≤0.01%, and the risk score for trisomy 18 was ≤0.01% in 295 cases, 0.04% in 1 case, and 0.23% in 1 case. Therefore, the sensitivity for detecting trisomy 21 was 100% (50/50 cases); the sensitivity for trisomy 18 was 98% (49/50 cases), and the specificity was 100% (297/297 cases).
Conclusion
In this study, chromosome-selective sequencing of cell-free DNA separated all cases of trisomy 21 and 98% of trisomy 18 from euploid pregnancies.
Diagnosis of fetal aneuploidies relies on invasive testing by chorionic villous sampling or amniocentesis in pregnancies that are identified by screening to be at high risk for such aneuploidies. In the 1970s and 1980s, the main method of screening for aneuploidies was by maternal age, with a cutoff of 35 years to define the high-risk group. This was associated with a 5% screen-positive rate and a detection rate of trisomy 21 of 30%. In the late 1980s and 1990s, screening was provided by a combination of maternal age and serum biochemistry in the second trimester, which resulted in improvement of the detection rate to 50-70%, with the same 5% screen-positive rate. In the last 15 years, the emphasis of screening shifted to the first trimester, where a combination of maternal age, fetal nuchal translucency (NT) thickness, maternal serum-free β-human chorionic gonadotropin (β-hCG), and pregnancy-associated plasma protein-A (PAPP-A) could identify approximately 90% of fetuses with trisomy 21, 18, and 13. In specialist fetal medicine centers, the addition of other first-trimester sonographic markers, which include the nasal bone and Doppler blood flow in the ductus venosus, hepatic artery, and across the tricuspid valve, could improve the detection rate of aneuploidies >95% and could reduce the screen-positive rate to <3%.
For Editors’ Commentary, see Contents
See related editorial, page 269
Recently, noninvasive prenatal detection of fetal aneuploidies has been achieved by exploitation of the presence of cell-free DNA (cfDNA) in maternal plasma. In trisomy 21, compared with euploid pregnancies, the amount of chromosome 21 in maternal plasma is slightly higher than that of other chromosomes, because there are 3, rather than 2, copies of fetal chromosome 21. Massively parallel shotgun sequencing (MPSS), which can identify and quantify millions of DNA fragments, has now made it possible to detect the increment in chromosome 21 in the plasma of affected pregnancies. With this approach, trisomy 21 (and to a lesser extent trisomy 18) has been detected successfully noninvasively. Essentially, maternal plasma DNA molecules are sequenced, and the chromosomal origin of each molecule is identified by a comparison with the human genome. In trisomy 21 pregnancies, the number of molecules that are derived from chromosome 21, as a proportion of all sequenced molecules, is higher than in euploid pregnancies. However, this approach requires a significant amount of DNA sequencing, which can be costly and has a limited throughput. Because MPSS is not selective in the chromosomal origin of the sequenced DNA fragments, and chromosome 21 represents only approximately 1.5% of the human genome, it is necessary to sequence many millions of fragments to ensure sufficient chromosome 21 counts. An alternative to MPSS that may overcome these limitations is selective sequencing of loci from only chromosomes under investigation. Such chromosome-selective sequencing, referred to as digital analysis of selected regions (DANSR), has been applied successfully to the noninvasive detection of trisomy 21 and 18. Sparks et al have introduced the fetal-fraction optimized risk of trisomy evaluation (FORTE) by extending the process of chromosome-selective sequencing to assay nonpolymorphic and polymorphic loci, where fetal alleles differ from maternal alleles, which enables the simultaneous determination of chromosome proportion and fetal fraction.
The objective of this study was to assess the prenatal detection rate of trisomy 21 and 18 and false-positive rate at 11-13 weeks’ gestation by the DANSR assay and the FORTE algorithm.
Materials and Methods
Study population
This was a nested case-control study of stored maternal plasma from 400 singleton pregnancies at 11-13 weeks’ gestation, including 300 pregnancies with euploid fetuses, 50 pregnancies with trisomy 21, and 50 pregnancies with trisomy 18. In all cases fetal karyotyping was carried out by chorionic villous sampling in our tertiary referral center, because screening by the combined test in the patients’ hospitals demonstrated that the risk for aneuploidies was >1 in 300. Gestational age was determined from the measurement of the fetal crown-rump length. The measured NT was transformed into likelihood ratio for each trisomy with the use of the mixture model of NT distributions. The measured free β-hCG and PAPP-A were converted into a multiple of the median (MoM) for gestational age that was adjusted for maternal weight, racial origin, smoking status, method of conception, parity, and machine for the assays. The nasal bone was assessed as being present or absent; blood flow across the tricuspid valve was classified as normal or regurgitant, and blood flow in the ductus venosus was classified according to the a-wave as normal or reversed.
Maternal venous blood (10 mL) that was collected before chorionic villous sampling in ethylenediaminetetraacetic acid vacutainer tubes (Becton Dickinson UK Limited, Oxfordshire, UK) was processed within 15 minutes of collection and centrifuged at 2000 g for 10 minutes to separate plasma from packed cells and buffy coat (plasma 1) and subsequently at 16,000 g for 10 minutes to further separate cell debris (plasma 2). Plasma 1 and 2 (2 mL each) were divided into 0.5-mL aliquots in separate Eppendorf tubes that were labeled with a unique patient identifier and stored at −80°C until subsequent analysis. Written informed consent was obtained from the women who agreed to participate in the study, which was approved by the King’s College Hospital Ethics Committee.
We searched our database and selected 50 consecutive cases of trisomy 21 and 50 cases with trisomy 18 with 2 mL of available stored plasma 2, corresponding to 4 tubes of 0.5-mL aliquots per case. Each 1 of these 100 aneuploid cases was matched with 3 euploid control subjects for length of storage of their blood samples; none of the samples were previously thawed and refrozen. Maternal blood was collected between March 2006 and August 2011. We excluded pregnancies that were conceived by in vitro fertilization.
Laboratory analysis
Plasma samples (4 tubes of 0.5 mL per patient) from selected cases were sent overnight on dry ice from London to the laboratory of Aria Diagnostics, Inc, in San Jose, CA. The following information was provided to Aria Diagnostics for each case: patient-unique identifier, maternal age, gestational age, date of blood collection, and fetal sex but not fetal karyotype. Before evaluation for fetal trisomy, Aria Diagnostics, Inc, assessed each sample for volume, adequacy of labeling, and risk of contamination or sample mixing and informed us that 25 samples did not meet their acceptance criteria (in 8 cases, the total plasma volume after pooling of individual tubes was <2 mL; in 5 cases, the labels on the tubes did not match the patient identifier on the file that was provided to the laboratory, and in 12 cases, there were potential issues of sample mixing or cross contamination after pooling of the individual tubes by laboratory personnel). In 11 cases, we had stored samples of plasma 1 (4 tubes of 0.5 mL per patient); however, in 14 cases, there was either no or insufficient plasma 1, which were replaced with the next available cases. The samples from these 25 cases were sent to Aria Diagnostics, Inc, and we were informed that all cases fulfilled the acceptance criteria of the laboratory. The 400 samples that fulfilled the acceptance criteria were then analyzed with their previously published technique of the DANSR assay with the FORTE algorithm.
Results were provided for the risk of trisomy 21 and 18 on each of the 400 cases that fulfilled the acceptance criteria, and the correlation was determined between the assay results with the fetal karyotype.
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