We show that fetal trisomy 21 could be detected in high risk pregnancies with high sensitivity and specificity by means of multiplexed sequencing of maternal plasma DNA. A key strength of the study is that the diagnostic performance was compared against full karyotyping, which is the gold standard for aneuploidy diagnosis. Another strength of this study is that the test performance was assessed based on a large number of trisomy 21 and unaffected pregnancies. In fact, this study reports the largest scale use of massively parallel genomic sequencing (so called next generation sequencing) for medical diagnostics to date and shows that it is practically feasible on a large scale.
Another strength of the study lies in the comparison of the diagnostic performance of sequencing protocols with two levels of sample throughput. The comparison allows us to deduce factors that are fundamental to the sensitive detection of fetal trisomy 21 and provide insight into ways for further improvements. Measurement of the percentage of plasma DNA that was from chromosome 21 was more precise with the use of the 2-plex protocol because seven times more plasma DNA molecules were analysed with this method than with the 8-plex protocol. All trisomy 21 fetuses, including those missed by the 8-plex protocol, were detected with the 2-plex protocol. In short, the diagnostic sensitivity of the sequencing approach improves with an increase in the number of plasma DNA molecules analysed per sample.18
There are some weaknesses in the study design. Our study was based on high risk pregnancies—those clinically indicated for amniocentesis or chorionic villus sampling—which ensured that full karyotyping results were available from each pregnancy. To investigate if the sequencing test might be useful for prenatal assessment of fetal trisomy 21 for all pregnancies in general, we determined the post-test probabilities for having a trisomy 21 fetus in women of different ages, and hence with different pretest risks. The data suggest that the sequencing test result can substantially alter the probabilities for having a trisomy 21 fetus even in women with low pretest risk.
Another potential weakness of the study was that an archived sample set was included. However, both the prospectively collected and archived maternal plasma samples were processed using the same protocols and analysed prospectively. There were also no significant differences in the fetal DNA concentrations and test performances between the two sample groups. In this study 5.6% of the collected maternal plasma samples were of compromised quality. Reportable sequencing results were not achieved for 11 samples. All these samples were identified by the predefined quality control steps. In routine practice, laboratory reports would not be issued for such samples, and another blood specimen would be requested.
We found other aneuploidies besides trisomy 21 among the recruited pregnant women. For this study, we focused on the diagnostic performance of the sequencing approach for fetal trisomy 21 because our previous data showed that the measurements of the proportion of DNA molecules from chromosomes 18 and 13 were much less precise.24 25
More research is required to develop protocols to improve the precision for measuring amount of DNA molecules from chromosomes 18 and 13.29
Our data reveal that the main value of the maternal plasma DNA sequencing test is to rule out fetal trisomy 21. Hence, with the current diagnostic performance, it is more suitable as a screening test to stratify pregnancies whose risk for trisomy 21 warrants the consideration of amniocentesis or chorionic villus sampling. In this study, we performed the sequencing test after a pregnancy was deemed to be at increased risk for trisomy 21 according to the current prenatal screening programmes. The false positive rates of the current screening programmes are about 5%, and all of these pregnanct women are offered the option of invasive testing. However, if we took into consideration the results of the sequencing test, trisomy 21 could be ruled out in 98% of those pregnancies. This would leave just 0.1% (that is, 5%×(100%−98%)×100%) of all pregnant women needing referrals for amniocentesis or chorionic villus sampling. Most of our studied pregnancies were in the first trimester, which suggests that it is possible to implement the test even in early pregnancy as a second tier screening test after the first trimester combined test, which is already in use in many parts of the world.
On the other hand, our post hoc analysis shows that the sequencing test result may alter clinical decisions even in women at low risk for trisomy 21. Hence, there is the potential to apply the sequencing test as a first tier screening test. However, massively parallel genomic sequencing is currently expensive. To determine its suitability as a first tier screening test would require formal investigation of both its diagnostic performance and cost benefit. We used the sequencing test in this study for detection of fetal trisomy 21, but, with some improvements in bioinformatics, it could be used to detect other aneuploidies, such as trisomy 18 and trisomy 13.29
Furthermore, our group has recently shown that when more sophisticated sequencing protocols and bioinformatics algorithms are applied to the analysis of maternal plasma DNA, it may be possible to perform a genetic and mutational scan across the whole genome of the fetus in a non-invasive manner.30
Thus, it is likely that maternal plasma DNA sequencing will play an increasingly important role in the future developments of prenatal screening and diagnosis.
What is already known on this topic
- Non-invasive prenatal detection of fetal trisomy 21 is achievable by massively parallel sequencing of maternal plasma DNA
- Its diagnostic performance and practical feasibility in the clinical setting has not been tested on a large scale
What this study adds
- Among high risk pregnancies clinically indicated for invasive prenatal diagnosis, non-invasive detection of fetal trisomy 21 can be achieved with the use of multiplexed massively parallel sequencing of maternal plasma DNA at 100% sensitivity and 97.9% specificity, giving a 96.6% positive predictive value and 100% negative predictive value
- The sequencing test could be used to rule out trisomy 21 among high risk pregnancies before proceeding to invasive diagnostic testing to reduce the number of cases requiring amniocentesis or chorionic villus sampling