A New Era in Prenatal Diagnosis: The Use of Cell-Free Fetal DNA in Maternal Circulation for Detection of Chromosomal Aneuploidies

Prenatal screening for chromosomal aneuploidies is a fundamental part of routine obstetric care in most countries. Typically, maternal age, weight, ethnicity, serum biomarkers (including pregnancy-associated plasma protein A, human chorionic gonadotropin, α-fetoprotein, inhibin A, and estriol), and sonographic features (i.e., nuchal translucency) are included in a risk algorithm to determine the probability of the fetus being affected. Pregnant women identified as at high risk according to the prenatal screen can then undergo invasive procedures, such as amniocentesis and chorionic villus sampling, to confirm the diagnosis. Current prenatal-screening methods are able to identify approximately 90% of pregnancies affected by trisomy 21 (Down syndrome) at a false-positive rate of approximately 5%. Given that the prevalence of chromosomal aneuploidies is generally quite low, a false-positive rate of 5% means that a large number of women with unaffected pregnancies undergo invasive procedures, putting the fetus at an unnecessary risk for miscarriage. The discovery of fetal cell-free DNA in the plasma of pregnant women 14 years ago opened up the possibility of identifying chromosomal abnormalities noninvasively, through a single blood sample. Approximately 10% of cell-free DNA in the maternal circulation is of fetal origin, and this property was initially exploited to determine rhesus D status and the sex of the unborn fetus. The advent of next-generation DNA sequencing, however, has allowed prenatal detection of chromosomal aneuploidies, including trisomy 21, from maternal blood. In brief, the proportion of chromosome 21 DNA molecules in maternal plasma is measured directly; an increase above a predetermined threshold is indicative of trisomy 21. The clinical performance of this noninvasive prenatal test has been promising, with recent clinical studies having shown a diagnostic sensitivity of 100% and a diagnostic specificity of 98%–99%, compared with full karyotyping by invasive means. When used as a second-tier screening procedure, this technology also has the potential to markedly reduce the number of women undergoing invasive diagnostic procedures, which produces considerable cost savings. In this article, 4 leaders in the field of noninvasive prenatal diagnosis provide their opinion on this exciting advancement. Can you briefly describe how next-generation sequencing (NGS)7 has been applied to the detection of chromosomal aneuploidies with cell-free fetal DNA? Are there any other laboratory techniques that have been explored for this purpose? Barry Hoffman: NGS noninvasively detects chromosomal aneuploidy of the fetus prenatally by determining the relative proportion of DNA from the affected chromosome in the cell-free fragmented DNA circulating in the maternal blood. Many millions of sequencing reads at an appropriate depth of coverage are required to reliably quantify the miniscule increment or decrement in chromosomal dosage in the maternal circulation due to the fetal aneuploidy, and this has become feasible only with the advent of NGS technology. Either all of the circulating DNA fragments are sequenced without preselection by using massively parallel shotgun sequencing (MPSS) in a nontargeted approach, or DNA fragments from the target of interest, in this case the aneuploidic chromosome along with one other, are first selected before subsequent sequencing. The latter requires considerably less sequencing, thereby improving throughput and decreasing reagent cost, but is inherently more complex by requiring a strategy to select DNA from targeted regions. Some of the methods correct for the fraction of cell-free DNA in the maternal circulation that originates from the fetus, as this influences the magnitude of aneuploidic chromosomal dosage observed against the backdrop of the much larger maternal disomic contribution. Typically, the fetal fraction is of the order of 10%, but results that are sufficiently reliable can be obtained when the fraction is as low as 3%–4%. Another approach is to enrich the fetal fraction, for example by exploiting methylation differences between fetal and maternal DNA, before sequencing to increase the fetal signal. Dennis Lo: Most published studies on the use of NGS for detecting chromosomal aneuploidies are based on random, or shotgun, sequencing. When mapped back to the reference human genome, the sequencing data yield a proportional representation of each chromosome in maternal plasma. When used to detect a fetal trisomy, the aneuploid chromosome would show an increased representation in maternal plasma in the presence of a fetus suffering from the trisomy. Apart from random sequencing, a number of researchers have published on approaches based on targeted sequencing, in which sequences from the chromosomes of interest are selectively captured or amplified and then sequenced. Apart from NGS, a number of reports have studied the use of the allelic ratio of plasma RNA and DNA methylation markers for the noninvasive prenatal detection of fetal chromosomal aneuploidies. In my opinion, these methods are not as mature as strategies based on NGS, especially in terms of robustness and population coverage. Jacob Canick: Professor Lo's hallmark finding in 1997, that DNA fragments of fetal as well as maternal origin are present in the circulation of pregnant women, provided the biological basis for developing an NGS strategy for fetal-aneuploidy detection. Simultaneous sequencing of millions of such DNA fragments allows for computerized identification of the chromosome of origin for each fragment. Therefore, the ability to discern a fetal trisomy on a BACKGROUND of cell-free DNA from a euploid mother is really an exercise in analytical imprecision. A fetus affected by a particular trisomy will have a 50% increase in that chromosome's contribution to the total fragments sequenced, but the increase is diluted according to the proportion of fetal DNA present in the maternal plasma sample. The actual increase caused by the trisomic fetus will be, on average, one half of whatever the percent fetal DNA contribution is in that sample. Other molecular methods that have been examined previously with some success involve epigenetic differences between fetal and maternal DNA (e.g., methylation) and RNA (e.g., fetal-specific vs maternal-specific gene transcription). However, the degree of epigenetic variation is different in different populations, so that so-called universal coverage by using such methods is difficult to attain. Dirk van den Boom: The key questions after Dennis Lo's 1997 publication related to (A) choosing the most appropriate analyte (DNA, RNA) to enable commercially viable sample-collection procedures; (B) maximizing the yield of the chosen analyte in nucleic acid extraction procedures; and (C) identifying the most appropriate technology to reliably detect the fetal aneuploidy in the chosen analyte given that the majority of information in maternal blood/ plasma is maternally derived. A variety of methods were initially researched, among them RNA-based methods, methods relying on epigenetic differences between the maternal and fetal genome, and different detection technologies, including digital PCR, mass spectrometry, and sequencing. NGS is currently the favored and dominant detection technology, because it provides the necessary amount of data points that allows for accurate detection of a fetal trisomy. At the Sequenom Center of Molecular Medicine, we have chosen to implement a whole-genome approach towards fetal aneuploidy detection by using NGS. DNA is extracted from maternal plasma. Most of the extracted DNA is apoptotic in nature and highly fragmented into relatively short continuous stretches (around 150 bp). Then, a DNA library representative of the maternal and fetal genomes is prepared from these fragments. Following amplification, several million DNA fragments of the library are sequenced in a massively parallel fashion. For each component of the library, the process generates 36 bases of continuous sequence information (sequence read), sufficient to uniquely identify the chromosomal origin of the sequence/fragment. After completion of the sequencing process, all sequence reads generated per patient sample are mapped back to their chromosomal location on the human genome, and the of fragments that with each chromosome is these data in one can the relative representation of each chromosome vs a of reference The with the of the chromosome and is a and because it is based on a large number of of markers of data points per with each sequence being an At this the detection of a fetal trisomy, such as trisomy 21, The cell-free DNA from the plasma of a pregnant women a fetus with trisomy 21 will more fragments from chromosome 21, due to the of that chromosome in the fetal and in the cell-free fetal DNA present in maternal plasma. In the sequencing process this will to an of chromosome 21 sequences relative to a of reference a trisomy 21 is identified as a increase in the chromosome 21 representation compared to plasma from pregnant women a euploid fetus. case for trisomy 21 can be to the identification of or in the chromosome representation of any the laboratory test currently by the Sequenom Center of Molecular currently on chromosomes 21, and chosen whole-genome approach is so that fetal aneuploidies can be and without to the test as clinical are and an of the cost of NGS this technology to a noninvasive molecular with a and performance to invasive different NGS been for the of noninvasive prenatal Are there Barry Hoffman: A number of NGS have been for noninvasive prenatal including from and the DNA by or PCR, before sequencing, which has the of that be The first use by DNA the latter by DNA The to the DNA by the or are identified by the to the In the case of the the 4 are one at a and the that is when one is is The other into the DNA to determine the these published studies have shown that all are of trisomy 21 on the can the of the and to determine the will reduce reagent and the of the thereby the of NGS to clinical Dennis Lo: most have the use of the are also a of the use of the The results generated by using such are generally However, the of the sequencing and the rate and the sequencing results would clinical (e.g., and maternal for the use of amplification, has been For the detection of chromosomal aneuploidies chromosomes to this (e.g., chromosomes and such are and have been shown to be recent the use of a that not an for maternal plasma a not to be a would be to test a number of sequencing (e.g., for this Jacob Canick: my NGS have been and for this The most has been by using the on the in in which was by using the from provided that this also be Dirk van den Boom: there are a of other NGS such as the or the we to implement the test on the The for this were the high data and of the technology at the of for the clinical laboratory sample and cost were also key in of for is the for this of this test be this to for Barry Hoffman: The of commercially that detect trisomy are on the order of the to by in the first or of NGS throughput on how well the laboratory has and from to the required to a clinical library sequencing, data and Other that on the throughput the of the sequencing technology the number of that can be in a single and the and of the sequencing reads that in on the required depth of coverage and with the the and relative amount of fetal DNA in the circulating cell-free DNA of the mother is in published studies have shown that can reliably detect fetal as as the of in the first with of and the of a routine first of the in which the fetus is for for and for the of Dennis Lo: The is approximately The test can be from of is highly with for maternal For pregnant women can first be by for of nuchal of their and as high risk by such can then be to undergo the 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been less The also that because the test performance in that the performance would be in a population undergoing In the first was published that the performance of screening for trisomy 21 and trisomy by using sequencing in a routine population of The that of fetal cell-free DNA in the maternal plasma of a population was as as previously in A number of other studies the performance of the test in a routine population of women undergoing screening for aneuploidy are currently with results the the test for will the of women and to the of circulating cell-free fetal DNA in the maternal circulation as the approach of aneuploidy prenatal Dennis Lo: The of the of fetal aneuploidy using maternal plasma DNA sequencing is the of fetal DNA in maternal plasma. as there is that women as high risk by screening have a different fetal DNA that in that the NGS test have the performance in The is more an as there are more women with the in the cost of sequencing and such will become less of an that in the to maternal plasma DNA for aneuploidy screening will become a Jacob Canick: The of screening to all pregnant women be by there a basis to different performance in the population compared to the and are there more to before have examined there are any to this of that a number of which are related to the of differences in and and the has been For the for being high risk not the fetal fraction or the test that is for trisomy 21 or euploid In markers that are currently used (i.e., the serum markers and the fetal nuchal translucency) are not or are with fetal fraction or the more for not using NGS to test the population are cost and and Dirk van den Boom: The of NGS has been in pregnant women at high risk for fetal women of maternal age, of fetal aneuploidy, results or In to the the of and and the for a opinion that cell-free fetal DNA be to at increased risk of The also an and that the use of cell-free fetal DNA as a prenatal screening test for fetal aneuploidy for trisomy 21 and in women all for and in with a targeted approach with NGS for detection of fetal trisomy has been included a sample of women, and more clinical will be required before such a test be generally applied in a screening At this currently serum approaches are more for the their identify currently not by cell-free fetal this technology been applied to other chromosomal aneuploidies from trisomy from single Barry Hoffman: studies have the potential of NGS to detect a of molecular in fetal circulating cell-free DNA, including chromosomal aneuploidies, and number The technology be to identify each of with such as of sequenced and of on the and of the is that NGS will in the detection of fetal and there is to the of sequencing of reliably detecting all of DNA molecular and in a single the technology has been applied commercially to the detection of sex chromosome aneuploidy, such as and and to the of Dennis Lo: In to trisomy 21, the technology has been applied to and as well as are also data that the sequencing test can be used to detect caused by as well as chromosomal For the the depth of sequencing would to be have shown that the sequencing approach can be used for the noninvasive prenatal diagnosis of (e.g., including caused by For such a targeted sequencing approach to be more Jacob Canick: is to that trisomy 21, the most aneuploidy at also to be the of the aneuploidies to identify by NGS of maternal plasma and and have been more difficult to identify with the of performance as for trisomy 21, but the performance for aneuploidies is sufficiently high to clinical use of NGS to test for However, test results for any of these aneuploidies, including trisomy 21, are not sufficient to a diagnosis. diagnostic is for all NGS results to that false-positive euploid are Dirk van den Boom: The for the test included with fetal trisomy and in to trisomy 21. were with high diagnostic technology for fetal aneuploidy detection an approach that in can the of fetal other 21, or become for 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fraction with the component being of maternal In pregnancies only one of the is the of the chromosome is as as the fetal fraction is above the detection The fetus only a amount to the euploid maternal have on with the test can be applied to in The of the chromosomal representation the noninvasive detection of fetal trisomy in maternal in is not by In opinion, you this test as a diagnostic test for prenatal screening and the maternal serum screen will it be an to screening Barry Hoffman: is that of fetal cell-free DNA in maternal plasma is a test the trisomy screen that and However, the is also considerably more can be that can to for the molecular or through will it as their However, it is that prenatal-screening will have the to for the universal of molecular to all women undergoing prenatal A to this is screening is first and molecular only to the screen The performance of such screening approaches to that of molecular and the to a be due is to the to the of In has shown that screening is cost when the rate of the screen is to 5%. 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