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Recent progress in non-invasive prenatal diagnosis
Seminars in Fetal & Neonatal Medicine (2008) 13, 57e62
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/siny
Recent progress in non-invasive prenatal diagnosis Sinuhe Hahn*, Xiao Yan Zhong, Wolfgang Holzgreve University Women’s Hospital/Department of Biomedicine, University Hospital Basel, Switzerland
KEYWORDS Cell-free fetal DNA/mRNA; Down syndrome; Fetal cells; Maternal blood; Non-invasive; Prenatal diagnosis
Summary Although the first finding that fetal cells can enter the maternal circulation was made more than a century ago, it is still unclear if this finding will be translated into a clinically useful diagnostic tool in the foreseeable future. However, significant progress has been made via the analysis of cell-free fetal DNA in maternal plasma/serum and clinical services are now already being offered for the determination of fetal rhesus D status and sex. Currently, however, this technology is really only suited for the analysis of fetal genetic loci completely absent from the maternal genome. The detection of more subtle fetal genetic traits, such as point mutations involved in Mendelian disorders (thalassaemia, cystic fibrosis), is considerably more complex. Preliminary reports indicate that the detection of fetal aneuploidies might be possible using epigenetically modified genes, e.g. maspin on chromosome 18. Additionally, an exiting recent development is that it might be feasible to detect Down syndrome via the quantitative assessment of placentally derived cell-free mRNA of chromosome-21-specific genes such as PLAC4. ª 2007 Elsevier Ltd. All rights reserved.
Current status After more than three decades of intensive research, the non-invasive analysis of fetal genetic traits via the examination of a simple maternal blood sample has become a clinical reality, and is already being used by several European centres for the determination of fetal sex and of rhesus D (RHD) status.1e3 These analyses are relatively simple to perform because they involve the detection of fetal genes completely absent from the maternal genome and, as such, can be performed with high degrees of accuracy using appropriate polymerase chain reaction (PCR) strategies. * Corresponding author at: Laboratory for Prenatal Medicine and Gynecological Oncology, University Women’s Hospital/Department of Research, Hebelstrasse 20, CH4031 Basel, Switzerland. Tel.: þ416 1265 9224; fax: þ416 1265 9399. E-mail address: [email protected] (S. Hahn).
Although this clinical breakthrough is to be lauded, the real challenge still lies ahead: namely, the devising of methods or approaches that will permit the efficacious detection of fetal single-gene disorders (e.g. b-thalassaemia, cystic fibrosis) or fetal chromosomal anomalies (e.g. trisomy 21/ Down syndrome). These analyses are considerably more difficult to perform because they involve fetal genetic changes that differ only slightly from the maternal genome, i.e. point mutations or the addition of a single chromosome, frequently of maternal origin. New strategies are needed to meet these challenges, the most promising of which are highlighted in this chapter.
Fetal cells versus cell-free DNA Two strategies have emerged for the non-invasive analysis of fetal genetic traits: (1) the isolation of fetal cells that have entered the maternal circulation; and (2) the
1744-165X/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.siny.2007.11.001
58 analysis of placentally derived cell-free fetal DNA (cff-DNA).1e3 The main advantage of isolated fetal cells is that they offer a pure source of the entire fetal genome, without the possible inclusion of any maternal genetic material.1 This is important when examining Mendelian disorders, as the fetus will have inherited one copy of the mutant gene from the mother and the other from the father, especially in those constellations where mother and father have the same mutation. This essential condition of having access to pure fetal genetic material cannot currently be met via the analysis of cff-DNA because of the high preponderance of maternal cell-free DNA (cf-DNA) sequences in the maternal plasma samples.1 Therefore, it is currently not possible to determine whether a fetus has inherited a maternal mutation via the analysis of cff-DNA. Indeed, simply determining the presence of a paternal mutation different to that of the mother is challenging via this approach. This is because it is frequently necessary to resort to the use of sophisticated approaches for such analyses, e.g. the size-fractionation of the cf-DNA sample or the use of expensive mass spectrometric devices. In theory, the use of fetal cells enriched by maternal blood should offer a simple and cost-effective method for the prenatal detection of fetal aneuploidies by fluorescence in-situ hybridization (FISH) analysis, especially when using automated microscope systems, which would robotically identify and analyse putative target fetal cells.1 However, although these premises sound attractive, it should be noted that the enrichment of fetal cells from maternal blood has been pursued for 30 years and in this time no clinical application has come to fruition. This is not likely to change in the near future.1 The major problem that continues to beset the efficient use of fetal cells and to hinder any meaningful progress is their scarcity; they occur in the order of one fetal cell per ml of maternal blood. As it is not possible to examine one million maternal cells to detect a sole fetal cell, the only way to overcome this problem is by some form of enrichment, a process during which it is likely that a large number of these rare fetal cells are lost. This deficit was highlighted in the large-scale multicentre NIFTY study, conducted under the auspices of the US National Institutes for Health (NIH), in which around 3000 clinical cases were examined.4 It remains the largest study of its type to date and used magnetic cell sorting (MACS) or fluorescent activated cell sorting (FACS) approaches to recover fetal cells. The fetal cells could only be detected with sensitivities of the order of 50%.4 Thus, this large and well-designed study clearly illustrates that such, or analogous, approaches are not yet ready to meet the robust needs of everyday clinical life. A further problem is that although fetal cells can offer a pure source of fetal genetic material, the analysis of certain putative target fetal cells, such as erythroblasts, can be confounded by the presence of similar maternal cells.5 Consequently, a fair degree of controversy existed in the literature as to whether fetal erythroblasts were present in the maternal circulation. As it was important to clarify this before undertaking any further studies, the Basel laboratory examined single enriched erythroblasts
S. Hahn et al. that had been isolated by manual micromanipulation using finely-drawn glass needles.6 To determine the fetal or maternal origin of these cells, we used two fetal genetic markers that were completely absent from the maternal genome in the cases we examined: the Y chromosome and the rhesus D gene. Our study clearly showed that approximately 50% of the trafficking erythroblasts in maternal circulation were of fetal origin, a finding that highlights that care should be taken when examining putative target cells.6 The fact that this study of 19 cases remains that largest in the literature indicates that this approach is tedious and labour intensive. Despite these technical challenges, the ability to examine such isolated erythroblasts by PCR indicated that this approach might be useful for the analysis of fetal Mendelian disorders. As a model system, most of these studies focused on b-thalassaemia because of the high prevalence of this disorder in certain population groups and the large number of well-defined mutations. In the first of these studies, pools of putative fetal erythroblasts, identified on the basis of expression of embryonic globin molecules, were used for the prenatal determination of fetal thalassaemia.7 Pools of putative fetal cells were examined to overcome the problem of allele drop out (ADO). This can occur when examining single or small numbers of cells by PCR, whereby certain loci fail to be amplified efficiently, leading to the recording of erroneous results. Although this approach is sensible in this context, the presence of any maternal cell that has slipped into this pool will invariably lead to an incorrect analysis. For this reason, the second study to investigate the use of isolated fetal cells for the prenatal detection of bthalassaemia examined individually isolated erythroblasts by single-cell PCR, thereby eliminating the risk of any maternal contamination.8 In both of these studies, however, fewer than six cases were examined, making it very difficult to determine whether these were just scientifically interesting explorations or if they could lead to clinically useful applications. During this period, studies from the Basel laboratory clearly showed that erythroblasts e and especially those of fetal origin e exhibited very high rates of ADO, which could approach or exceed 50% of the examined alleles.9 Furthermore, these studies indicated that it would be necessary to examine four or five individual fetal erythroblasts to overcome this deficit and obtain clinically relevant degrees of accuracy. A further feature we observed in the Basel laboratory is that fetal erythroblasts are not amenable to analysis by FISH and that a large proportion are actually refractory to such analysis.10 This feature appears to be due to a peculiarly dense nucleus of apoptotic character, and appears to be triggered by the transition of these fetal cells from the relatively hypoxic fetal environment into the oxygen-rich maternal circulation. Consequently, any future studies using these cells will need to address these two aspects to ensure that the high degrees of accuracy required for prenatal diagnosis are attained. These aspects are being considered by the EU-funded SAFE (special advances in fetal evaluation) network (http://www.safenoe.org), which is analysing fetal cells via a two-pronged strategy. On the one hand, new enrichment methods, such as novel and more potent anti-CD71
Recent progress in non-invasive prenatal diagnosis antibodies,11 are being examined in a multi-laboratory manner. On the other hand, target cells other than the traditional fetal erythroblast are being analysed: considerable efforts are being expended on trafficking fetal stem cells and (because of the mixed results that have been achieved so far with trafficking fetal erythroblasts) on deported trophoblasts. Despite the pitfalls surrounding the analysis of fetal erythroblasts, efforts are still being expended on this cell type. These are largely being undertaken by the Graz and Athens groups. In this context, the Athens group (a member of SAFE) has continued to explore the use of isolated fetal erythroblasts in the prenatal determination of b-thalassaemia.12 The unique aspect of this study is that putative fetal erythroblasts were isolated by laser-mediated micromanipulation using a PALM Microlaser system. This recent study, performed in collaboration with the Graz centre (also a member of SAFE), indicated that this approach might be feasible. It also served to confirm observations made by the Basel group with regard to extraordinarily high rates of ADO.9 The advantage of isolated fetal cells with stem-cell-like characteristics is being pursued by the Perugia group of SAFE. It is thought that these cells might be amenable to cell culture.13 Pools of expanded cells could then be used both for complex molecular analysis for Mendelian disorders e greatly reducing the risk of ADO e and for opening up the possibility of obtaining a full karyotype by the use of specialized FISH approaches such as spectral karyotyping (SKY). Such a feature would represent a true breakthrough in non-invasive prenatal diagnostics as it would deliver the same degree of information currently attainable only via an invasive procedure. To optimize the efficacy of trophoblast enrichment14 and detection, novel markers are being sought by Guetta and colleagues in Israel via the application of cuttingedge proteomic approaches in collaboration with a number of other SAFE participants. Although cells of trophoblast origin have been reported to display mosaic characteristics, it is possible that this might not be a trait of all deported trophoblasts and, as such, the focus is being placed on cells that display normal karyoptypes. The second arm of the SAFE strategy is to optimize the automated recognition of rare fetal cells using systems developed by a number of small and medium enterprises (SMEs) participating in the SAFE network, such as PALM Microlaser, Metasystems and Imstar. The goal here is to develop systems that can e reliably and automatically e detect fetal cells from preparations of maternal blood. It is hoped that, by reducing the labour-intensive search for rare cells, these processes will contribute greatly in cost reduction, an important consideration when considering future clinical applications. Although future developments can be viewed with cautious enthusiasm, it should be clear to all involved that the main challenge remains: to have a system that permits the reliable and simple retrieval of rare fetal cells from maternal blood samples.1 This system will also have to be sufficiently cost-effective to compete with other developments, such as the analysis of cff-DNA/RNA, approaches that are readily amenable to automation. It remains to be determined whether these hurdles can be overcome.
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Non-invasive prenatal detection of Down syndrome: will it become a reality? The detection of fetal aneuploidies, particularly trisomy 21, is a major challenge in non-invasive prenatal diagnosis. As noted above, although initially promising, most examinations using fetal cells enriched from maternal blood have floundered due to the scarcity of these cells.1 The use of cffDNA for such an analysis is severely hampered by the fact that this fetal material represents only 3e5% of the total circulatory DNA in maternal plasma.15 This makes the quantitative determination of fetal loci, for instance short tandem repeats (STRs) or single nucleotide polymorphism (SNPs) that could be used to determine fetal aneuploidies very difficult, even when resorting to such approaches as size-fractionation of cff-DNA.15 Thus, given the current limitations in technology, an alternative means is needed. This alternative now seems to be provided by the presence of placentally derived cell-free fetal messenger RNA (cff-RNA) in maternal plasma; a salient discovery by the Hong Kong group.16 The main advantage of cff-RNA over cff-DNA is that it is possible to select for placenta-specific mRNA species not expressed by any maternal tissues. Hence, the analysis of cff-RNA can be viewed in the same way as the analysis of fetal genes completely absent from the maternal genome, as is currently used for fetal sex and RHD determinations. This implies that the analysis can simply focus on the fetal gene being examined and need not be concerned with any maternal background ‘‘noise’’. In its pivotal study, the Hong Kong group focused it attention on the PLAC4 (placenta specific 4) gene, as expression of this gene has previously been shown to be restricted to the placenta.16 Furthermore, this gene is important because it is located on chromosome 21 and, as such, a quantitative analysis of its expression might be useful in determining the ploidy of this important chromosome. The problem facing the investigators was that it was very clear from the outset that crude quantitative analysis of total PLAC4 mRNA levels would not yield sufficient information to accurately enumerate chromosome 21. To overcome this technical challenge, the group made use of its expertise with mass spectrometry, which had previously been shown to be useful for the detection of fetal point mutations. To enumerate chromosome 21 levels, the team made use of SNPs located in the PLAC4 gene. In those cases where the fetus was heterozygous for this SNP locus, the PLAC4 mRNA transcripts would, in cases of normal ploidy, contain equal ratios of each allele (1:1 ratio). If, however, an aneuploidy was present, then the ratio would shift to 2:1 (Fig. 1). Although small, this study is significant in that the analysis of ten cases with trisomy 21 demonstrated that fetal Down syndrome could be detected with a sensitivity of 90% and a specificity of 96%. This approach, then, appears to be very promising. The drawback of this approach, as currently used, is that it relies on the fetus being heterozygous for the SNP locus being interrogated. This constellation might be infrequent, particularly in certain ethnicities. Consequently, the number of SNP targets will need to be expanded and, if necessary, so will the number of chromosome-21-specific cff-RNA species.
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Heterozygous PLAC4 gene
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Placental transcription into mRNA
Heterozygous cff-RNA in maternal plasma
RT-PCR and MS analysis
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Quantitation of SNP allelic ratios by MS 1:1
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Figure 1 Schematic representation of the non-invasive detection of fetal Down syndrome via the analysis of placentally derived PLAC4 cf-RNA. cff-RNA, placentally derived cell-free fetal messenger RNA; mRNA, messenger RNA; MS, mass spectroscopy; RT-PCR, real-time polymerase chain reaction; SNP, single nucleotide polymorphism.
In a second study, Lo and colleagues attempted to overcome some of these issues by examining another technological breakthrough: digital PCR, a process whereby very small changes in template copy numbers can be accurately enumerated.17 The advantage of this technology over mass spectrometry is that it does not rely on the fetus being heterozygous for a particular SNP locus, i.e. measurements can be conducted if the fetus is also homozygous for the SNP locus in question. Not only would this greatly simplify the analysis, it would no longer be restricted to particular genetic constellations.
Trisomy 18: detectable via epigenetic modification of the maspin gene? Dennis Lo’s Hong Kong group was also instrumental in showing that epigenetically modified cff-DNA sequences might be suited for the detection of fetal aneuploidies. Epigenetic modifications are somatic alterations of the DNA that do not alter the genetic sequence but that do affect gene expression. A widely described form arises via the methylation of cytosine nucleotides at the so-called CpG islands. If a genetic sequence is heavily methylated, also termed hypermethylated, then it will tend not to be expressed. A classic example is the second female Xchromosome, where gene expression is largely shut down by epigenetic mechanisms. As epigenetic differences might exist between maternal and fetal cf-DNA, the Hong Kong group set out to identify genetic loci that differ in the methylation pattern between mother and fetus, the rationale being that it might be possible to exploit such differences and to distinguish
between fetal and maternal cf-DNA fragments that have the same nucleotide sequence. In this context, a clear different was noted between the fetal and maternal methylation status of the maspin gene: the promoter of the gene is not methylated in the placenta but is hypermethylated in maternal blood.18 These differences can be exploited by the use of a bisulfite-conversion step, whereby unmethylated cytosine is converted into uracil whereas the methylated cytosine is left unchanged, to generate fetal DNA sequences that can be readily discriminated from maternal ones, for instance by mass spectrometry (Fig. 2). In their initial investigations, Lo and colleagues were able to show that hypomethylated fetal maspin cff-DNA sequences could be detected and quantified reliably by realtime PCR, and that the behaviour of these molecules was very similar to more classic cff-DNA sequences, such as SRY. As the mapsin gene is located on chromosome 18, the group then set out to determine whether analysis of this locus could be used for the non-invasive detection of trisomy 18, for instance by a epigenetic allelic ration (EAR) approach.19 In this assay, two alleles are quantified relative to each other, again yielding a 1:1 ratio in cases with normal ploidy or a 1:2/2:1 ratio in aneuploid cases (Fig. 2). For their analysis, the group made use of a of a singlebase A/C variation in the promoter region of this gene. Although this approach is interesting, the authors indicate that it might be technically challenging, as they could not achieve a clear discrimination between euploid and aneuploid cases even when examining genomic DNA obtained from pertinent placentae. As such, it is not surprising that no clear assessment could be made regarding the analysis of maternal plasma samples, as only three samples were examined from trisomy 18 cases. An additional limit of this approach is that an absolute requirement is that the mother is homozygous C/C for the single-base A/C variation being investigated, as the presence of maternal A alleles will confound the assay. It is likely that the chief technical problem to be overcome is the aggressive nature of the bisulfite conversion, which can lead to an enormous destruction of input template. This event is crucial because of the very small numbers of cff-DNA sequences in a given maternal plasma sample, as any destruction of this valuable material will seriously hamper the subsequent analysis. Hence, a more optimal approach, which provides an effective conversion of unmethylated fetal cytosine sequences, will need to be sought. Alternatively, the focus could be placed on fetal sequences, which are hypermethylated whereas the corresponding maternal sequence is hypomethylated. This condition is met by the RASSF1A gene.20 Unfortunately, as this gene is located on chromosome 3, it will not prove to be useful for the detection of any of the most common fetal aneuploidies.
cff-DNA as a screening marker for fetal aneuploidies: a useful approach? One of the advantages of real-time PCR analyses of cff-DNA is that they offer a quantitative indication of the amount of fetal sequences present in a given sample.21 In this manner,
Recent progress in non-invasive prenatal diagnosis
Heterozygous maspin locus in placenta (unmethylated)
61
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Figure 2 Schematic representation of the non-invasive detection of fetal trisomy 18 via the analysis of epigenetically modified maspin sequences. cff-RNA, placentally derived cell-free fetal messenger RNA; MS, mass spectroscopy; PCR, polymerase chain reaction.
elevations in cff-DNA have been noted in pregnancies affected by preeclampsia, or those at risk of this disorder.22 In addition, discrete elevations have been noted in pregnancies with trisomy 21 fetuses, but not those with trisomy 18.23,24 These findings have suggested that cff-DNA could serve as a screening marker to detect pregnancies at risk for such fetal anomalies. Although this approach might be worth considering, it should be noted that cff-DNA levels can only be reliably quantified using multi-copy sequences on the Y chromosome, such as the DYS14 locus.25 As such, these analyses are restricted to cases in which the fetus is male. Although epigenetically modified genetic loci such as the RASSF1A gene could serve as fetal gender-independent markers, it is currently unclear how precise the determination of fetal cfDNA levels via the analysis of this sequence is. It should also be noted that these analyses are costly and so perhaps might be too expensive to be considered as a screening tool.
Discussion of future developments Although certain fetal genetic traits can now be determined non-invasively with great confidence, and although promising approaches appear to be on the horizon, a lot more work is required before the determination of complex Mendelian disorders or fetal aneuploidies becomes a clinical reality. The use of fetal cells enriched from maternal blood might, in future, be facilitated by the combined use of new markers and optimized microscopic platforms that permit
their automated detection. However, even if this were to become a reality, it would probably not be suitable for high-throughput analysis of large sample numbers; this feature would be possible via the analysis of cff-DNA or cff-RNA as all the required steps are highly amenable to automation. Several developments are necessary for this occur, including the use of mass spectrometric instruments such as the MassArray, marketed by Sequenom. This equipment needs to be optimized for a routine clinical environment, as certain steps (such as the extension and purification of PCR products that form the basis this analysis) are not suitable for rigorous day-to-day diagnostic use. Despite this premise, it is highly likely that this tool will become an important part of future cff-DNA analyses. In addition, the preparation of samples for cff-RNA or epigenetically modified fetal cf-DNA sequences needs to be improved and made more efficient to ensure that sufficient fetal template molecules are obtained for the subsequent PCR analysis. The highlight of recent studies is certainly cff-RNA, which indicates that this approach might be useful in determining fetal aneuploidies. However, it should be noted that this study was performed on a very small number of samples (10 cases and 56 controls) and, hence, these results need to be verified in a large multi-centre study, perhaps in a similar setting such as NIFTY or SAFE. Also still unclear is the number of mRNA transcripts and SNP loci that need to be targeted, and whether the use of digital PCRbased approaches will render these concerns superfluous.
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Practice points Invasive prenatal diagnosis is associated with increased risk of fetal loss. Non-invasive analysis of fetal genetic traits is already being used for fetal sex and RHD determination. Fetal Down syndrome could be detected with a sensitivity of 90% and a specificity of 96% by a non-invasive procedure via the analysis of cffRNA. Trisomy 18 might be detectable via the analysis of epigenetic modifications of the maspin gene, but the applicability is very limited. The quantitative use of cff-DNA levels is limited by the current need for high copy sequences such as DYS14.
Research agenda Increase the quality and quantity of fetal genetic materials in maternal circulation for analysis. Optimize approaches (such as mass spectrometric analysis) for sensitive, specific and quantitative detection of fetal loci, which can used for the determination of aneuploidies. Perform large multi-centre studies.
References 1. Hahn S, Holzgreve W. Prenatal diagnosis using fetal cells and cell-free fetal DNA in maternal blood: what is currently feasible? Clin Obstet Gynecol 2002;45:649e56. 2. Bianchi DW, Avent ND, Costa JM, van der Schoot CE. Noninvasive prenatal diagnosis of fetal Rhesus D: ready for Prime(r) Time. Obstet Gynecol 2005;106:841e4. 3. Chiu RW, Lo YM. The biology and diagnostic applications of fetal DNA and RNA in maternal plasma. Curr Top Dev Biol 2004;61:81e111. 4. Bianchi DW, Simpson JL, Jackson LG, et al. Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. National Institute of Child Health and Development Fetal Cell Isolation Study. Prenat Diagn 2002;22:609e15. 5. Hahn S, Sant R, Holzgreve W. Fetal cells in maternal blood: current and future perspectives. Mol Hum Reprod 1998;4:515e21. 6. Troeger C, Zhong XY, Burgemeister R, et al. Approximately half of the erythroblasts in maternal blood are of fetal origin. Mol Hum Reprod 1999;5:1162e5. 7. Cheung MC, Goldberg JD, Kan YW. Prenatal diagnosis of sickle cell anaemia and thalassaemia by analysis of fetal cells in maternal blood. Nat Genet 1996;14:264e8.
8. Di NE, Ghezzi F, Vitucci A, et al. Prenatal diagnosis of betathalassaemia using fetal erythroblasts enriched from maternal blood by a novel gradient. Mol Hum Reprod 2000;6:571e4. 9. Garvin AM, Holzgreve W, Hahn S. Highly accurate analysis of heterozygous loci by single cell PCR. Nucleic Acids Res 1998; 26:3468e72. 10. Babochkina T, Mergenthaler S, De NG, et al. Numerous erythroblasts in maternal blood are impervious to fluorescent in situ hybridization analysis, a feature related to a dense compact nucleus with apoptotic character. Haematologica 2005;90: 740e5. 11. Prieto B, Candenas M, Ladenson JH, Alvarez FV. Comparison of different CD71 monoclonal antibodies for enrichment of fetal cells from maternal blood. Clin Chem Lab Med 2002;40: 126e31. 12. Kolialexi A, Vrettou C, Traeger-Synodinos J, et al. Noninvasive prenatal diagnosis of beta-thalassaemia using individual fetal erythroblasts isolated from maternal blood after enrichment. Prenat Diagn 2007;27:1228e32. 13. Coata G, Tilesi F, Fizzotti M, et al. Prenatal diagnosis of genetic abnormalities using fetal CD34þ stem cells in maternal circulation and evidence they do not affect diagnosis in later pregnancies. Stem Cells 2001;19:534e42. 14. Guetta E, Gutstein-Abo L, Barkai G. Trophoblasts isolated from the maternal circulation: in vitro expansion and potential application in non-invasive prenatal diagnosis. J Histochem Cytochem 2005;53:337e9. 15. Lo YM, Chiu RW. Prenatal diagnosis: progress through plasma nucleic acids. Nat Rev Genet 2007;8:71e7. 16. Lo YM, Tsui NB, Chiu RW, et al. Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection. Nat Med 2007;13:218e23. 17. Lo YM, Lun FM, Chan KC, et al. Digital PCR for the molecular detection of fetal chromosomal aneuploidy. Proc Natl Acad Sci U S A 2007;104:13116e21. 18. Chim SS, Tong YK, Chiu RW, et al. Detection of the placental epigenetic signature of the maspin gene in maternal plasma. Proc Natl Acad Sci U S A 2005;102:14753e8. 19. Tong YK, Ding C, Chiu RW, et al. Noninvasive prenatal detection of fetal trisomy 18 by epigenetic allelic ratio analysis in maternal plasma: theoretical and empirical considerations. Clin Chem 2006;52:2194e202. 20. Chan KC, Ding C, Gerovassili A, et al. Hypermethylated RASSF1A in maternal plasma: A universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis. Clin Chem 2006;52:2211e8. 21. Lo YM, Tein MS, Lau TK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62: 768e75. 22. Hahn S, Holzgreve W. Fetal cells and cell-free fetal DNA in maternal blood: new insights into pre-eclampsia. Hum Reprod Update 2002;8:501e8. 23. Lo YM, Lau TK, Zhang J, et al. Increased fetal DNA concentrations in the plasma of pregnant women carrying fetuses with trisomy 21. Clin Chem 1999;45:1747e51. 24. Zhong XY, Burk MR, Troeger C, Jackson LR, Holzgreve W, Hahn S. Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses. Prenat Diagn 2000;20:795e8. 25. Zimmermann B, El-Sheikhah A, Nicolaides K, Holzgreve W, Hahn S. Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma. Clin Chem 2005;51: 1598e604.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/siny
Recent progress in non-invasive prenatal diagnosis Sinuhe Hahn*, Xiao Yan Zhong, Wolfgang Holzgreve University Women’s Hospital/Department of Biomedicine, University Hospital Basel, Switzerland
KEYWORDS Cell-free fetal DNA/mRNA; Down syndrome; Fetal cells; Maternal blood; Non-invasive; Prenatal diagnosis
Summary Although the first finding that fetal cells can enter the maternal circulation was made more than a century ago, it is still unclear if this finding will be translated into a clinically useful diagnostic tool in the foreseeable future. However, significant progress has been made via the analysis of cell-free fetal DNA in maternal plasma/serum and clinical services are now already being offered for the determination of fetal rhesus D status and sex. Currently, however, this technology is really only suited for the analysis of fetal genetic loci completely absent from the maternal genome. The detection of more subtle fetal genetic traits, such as point mutations involved in Mendelian disorders (thalassaemia, cystic fibrosis), is considerably more complex. Preliminary reports indicate that the detection of fetal aneuploidies might be possible using epigenetically modified genes, e.g. maspin on chromosome 18. Additionally, an exiting recent development is that it might be feasible to detect Down syndrome via the quantitative assessment of placentally derived cell-free mRNA of chromosome-21-specific genes such as PLAC4. ª 2007 Elsevier Ltd. All rights reserved.
Current status After more than three decades of intensive research, the non-invasive analysis of fetal genetic traits via the examination of a simple maternal blood sample has become a clinical reality, and is already being used by several European centres for the determination of fetal sex and of rhesus D (RHD) status.1e3 These analyses are relatively simple to perform because they involve the detection of fetal genes completely absent from the maternal genome and, as such, can be performed with high degrees of accuracy using appropriate polymerase chain reaction (PCR) strategies. * Corresponding author at: Laboratory for Prenatal Medicine and Gynecological Oncology, University Women’s Hospital/Department of Research, Hebelstrasse 20, CH4031 Basel, Switzerland. Tel.: þ416 1265 9224; fax: þ416 1265 9399. E-mail address: [email protected] (S. Hahn).
Although this clinical breakthrough is to be lauded, the real challenge still lies ahead: namely, the devising of methods or approaches that will permit the efficacious detection of fetal single-gene disorders (e.g. b-thalassaemia, cystic fibrosis) or fetal chromosomal anomalies (e.g. trisomy 21/ Down syndrome). These analyses are considerably more difficult to perform because they involve fetal genetic changes that differ only slightly from the maternal genome, i.e. point mutations or the addition of a single chromosome, frequently of maternal origin. New strategies are needed to meet these challenges, the most promising of which are highlighted in this chapter.
Fetal cells versus cell-free DNA Two strategies have emerged for the non-invasive analysis of fetal genetic traits: (1) the isolation of fetal cells that have entered the maternal circulation; and (2) the
1744-165X/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.siny.2007.11.001
58 analysis of placentally derived cell-free fetal DNA (cff-DNA).1e3 The main advantage of isolated fetal cells is that they offer a pure source of the entire fetal genome, without the possible inclusion of any maternal genetic material.1 This is important when examining Mendelian disorders, as the fetus will have inherited one copy of the mutant gene from the mother and the other from the father, especially in those constellations where mother and father have the same mutation. This essential condition of having access to pure fetal genetic material cannot currently be met via the analysis of cff-DNA because of the high preponderance of maternal cell-free DNA (cf-DNA) sequences in the maternal plasma samples.1 Therefore, it is currently not possible to determine whether a fetus has inherited a maternal mutation via the analysis of cff-DNA. Indeed, simply determining the presence of a paternal mutation different to that of the mother is challenging via this approach. This is because it is frequently necessary to resort to the use of sophisticated approaches for such analyses, e.g. the size-fractionation of the cf-DNA sample or the use of expensive mass spectrometric devices. In theory, the use of fetal cells enriched by maternal blood should offer a simple and cost-effective method for the prenatal detection of fetal aneuploidies by fluorescence in-situ hybridization (FISH) analysis, especially when using automated microscope systems, which would robotically identify and analyse putative target fetal cells.1 However, although these premises sound attractive, it should be noted that the enrichment of fetal cells from maternal blood has been pursued for 30 years and in this time no clinical application has come to fruition. This is not likely to change in the near future.1 The major problem that continues to beset the efficient use of fetal cells and to hinder any meaningful progress is their scarcity; they occur in the order of one fetal cell per ml of maternal blood. As it is not possible to examine one million maternal cells to detect a sole fetal cell, the only way to overcome this problem is by some form of enrichment, a process during which it is likely that a large number of these rare fetal cells are lost. This deficit was highlighted in the large-scale multicentre NIFTY study, conducted under the auspices of the US National Institutes for Health (NIH), in which around 3000 clinical cases were examined.4 It remains the largest study of its type to date and used magnetic cell sorting (MACS) or fluorescent activated cell sorting (FACS) approaches to recover fetal cells. The fetal cells could only be detected with sensitivities of the order of 50%.4 Thus, this large and well-designed study clearly illustrates that such, or analogous, approaches are not yet ready to meet the robust needs of everyday clinical life. A further problem is that although fetal cells can offer a pure source of fetal genetic material, the analysis of certain putative target fetal cells, such as erythroblasts, can be confounded by the presence of similar maternal cells.5 Consequently, a fair degree of controversy existed in the literature as to whether fetal erythroblasts were present in the maternal circulation. As it was important to clarify this before undertaking any further studies, the Basel laboratory examined single enriched erythroblasts
S. Hahn et al. that had been isolated by manual micromanipulation using finely-drawn glass needles.6 To determine the fetal or maternal origin of these cells, we used two fetal genetic markers that were completely absent from the maternal genome in the cases we examined: the Y chromosome and the rhesus D gene. Our study clearly showed that approximately 50% of the trafficking erythroblasts in maternal circulation were of fetal origin, a finding that highlights that care should be taken when examining putative target cells.6 The fact that this study of 19 cases remains that largest in the literature indicates that this approach is tedious and labour intensive. Despite these technical challenges, the ability to examine such isolated erythroblasts by PCR indicated that this approach might be useful for the analysis of fetal Mendelian disorders. As a model system, most of these studies focused on b-thalassaemia because of the high prevalence of this disorder in certain population groups and the large number of well-defined mutations. In the first of these studies, pools of putative fetal erythroblasts, identified on the basis of expression of embryonic globin molecules, were used for the prenatal determination of fetal thalassaemia.7 Pools of putative fetal cells were examined to overcome the problem of allele drop out (ADO). This can occur when examining single or small numbers of cells by PCR, whereby certain loci fail to be amplified efficiently, leading to the recording of erroneous results. Although this approach is sensible in this context, the presence of any maternal cell that has slipped into this pool will invariably lead to an incorrect analysis. For this reason, the second study to investigate the use of isolated fetal cells for the prenatal detection of bthalassaemia examined individually isolated erythroblasts by single-cell PCR, thereby eliminating the risk of any maternal contamination.8 In both of these studies, however, fewer than six cases were examined, making it very difficult to determine whether these were just scientifically interesting explorations or if they could lead to clinically useful applications. During this period, studies from the Basel laboratory clearly showed that erythroblasts e and especially those of fetal origin e exhibited very high rates of ADO, which could approach or exceed 50% of the examined alleles.9 Furthermore, these studies indicated that it would be necessary to examine four or five individual fetal erythroblasts to overcome this deficit and obtain clinically relevant degrees of accuracy. A further feature we observed in the Basel laboratory is that fetal erythroblasts are not amenable to analysis by FISH and that a large proportion are actually refractory to such analysis.10 This feature appears to be due to a peculiarly dense nucleus of apoptotic character, and appears to be triggered by the transition of these fetal cells from the relatively hypoxic fetal environment into the oxygen-rich maternal circulation. Consequently, any future studies using these cells will need to address these two aspects to ensure that the high degrees of accuracy required for prenatal diagnosis are attained. These aspects are being considered by the EU-funded SAFE (special advances in fetal evaluation) network (http://www.safenoe.org), which is analysing fetal cells via a two-pronged strategy. On the one hand, new enrichment methods, such as novel and more potent anti-CD71
Recent progress in non-invasive prenatal diagnosis antibodies,11 are being examined in a multi-laboratory manner. On the other hand, target cells other than the traditional fetal erythroblast are being analysed: considerable efforts are being expended on trafficking fetal stem cells and (because of the mixed results that have been achieved so far with trafficking fetal erythroblasts) on deported trophoblasts. Despite the pitfalls surrounding the analysis of fetal erythroblasts, efforts are still being expended on this cell type. These are largely being undertaken by the Graz and Athens groups. In this context, the Athens group (a member of SAFE) has continued to explore the use of isolated fetal erythroblasts in the prenatal determination of b-thalassaemia.12 The unique aspect of this study is that putative fetal erythroblasts were isolated by laser-mediated micromanipulation using a PALM Microlaser system. This recent study, performed in collaboration with the Graz centre (also a member of SAFE), indicated that this approach might be feasible. It also served to confirm observations made by the Basel group with regard to extraordinarily high rates of ADO.9 The advantage of isolated fetal cells with stem-cell-like characteristics is being pursued by the Perugia group of SAFE. It is thought that these cells might be amenable to cell culture.13 Pools of expanded cells could then be used both for complex molecular analysis for Mendelian disorders e greatly reducing the risk of ADO e and for opening up the possibility of obtaining a full karyotype by the use of specialized FISH approaches such as spectral karyotyping (SKY). Such a feature would represent a true breakthrough in non-invasive prenatal diagnostics as it would deliver the same degree of information currently attainable only via an invasive procedure. To optimize the efficacy of trophoblast enrichment14 and detection, novel markers are being sought by Guetta and colleagues in Israel via the application of cuttingedge proteomic approaches in collaboration with a number of other SAFE participants. Although cells of trophoblast origin have been reported to display mosaic characteristics, it is possible that this might not be a trait of all deported trophoblasts and, as such, the focus is being placed on cells that display normal karyoptypes. The second arm of the SAFE strategy is to optimize the automated recognition of rare fetal cells using systems developed by a number of small and medium enterprises (SMEs) participating in the SAFE network, such as PALM Microlaser, Metasystems and Imstar. The goal here is to develop systems that can e reliably and automatically e detect fetal cells from preparations of maternal blood. It is hoped that, by reducing the labour-intensive search for rare cells, these processes will contribute greatly in cost reduction, an important consideration when considering future clinical applications. Although future developments can be viewed with cautious enthusiasm, it should be clear to all involved that the main challenge remains: to have a system that permits the reliable and simple retrieval of rare fetal cells from maternal blood samples.1 This system will also have to be sufficiently cost-effective to compete with other developments, such as the analysis of cff-DNA/RNA, approaches that are readily amenable to automation. It remains to be determined whether these hurdles can be overcome.
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Non-invasive prenatal detection of Down syndrome: will it become a reality? The detection of fetal aneuploidies, particularly trisomy 21, is a major challenge in non-invasive prenatal diagnosis. As noted above, although initially promising, most examinations using fetal cells enriched from maternal blood have floundered due to the scarcity of these cells.1 The use of cffDNA for such an analysis is severely hampered by the fact that this fetal material represents only 3e5% of the total circulatory DNA in maternal plasma.15 This makes the quantitative determination of fetal loci, for instance short tandem repeats (STRs) or single nucleotide polymorphism (SNPs) that could be used to determine fetal aneuploidies very difficult, even when resorting to such approaches as size-fractionation of cff-DNA.15 Thus, given the current limitations in technology, an alternative means is needed. This alternative now seems to be provided by the presence of placentally derived cell-free fetal messenger RNA (cff-RNA) in maternal plasma; a salient discovery by the Hong Kong group.16 The main advantage of cff-RNA over cff-DNA is that it is possible to select for placenta-specific mRNA species not expressed by any maternal tissues. Hence, the analysis of cff-RNA can be viewed in the same way as the analysis of fetal genes completely absent from the maternal genome, as is currently used for fetal sex and RHD determinations. This implies that the analysis can simply focus on the fetal gene being examined and need not be concerned with any maternal background ‘‘noise’’. In its pivotal study, the Hong Kong group focused it attention on the PLAC4 (placenta specific 4) gene, as expression of this gene has previously been shown to be restricted to the placenta.16 Furthermore, this gene is important because it is located on chromosome 21 and, as such, a quantitative analysis of its expression might be useful in determining the ploidy of this important chromosome. The problem facing the investigators was that it was very clear from the outset that crude quantitative analysis of total PLAC4 mRNA levels would not yield sufficient information to accurately enumerate chromosome 21. To overcome this technical challenge, the group made use of its expertise with mass spectrometry, which had previously been shown to be useful for the detection of fetal point mutations. To enumerate chromosome 21 levels, the team made use of SNPs located in the PLAC4 gene. In those cases where the fetus was heterozygous for this SNP locus, the PLAC4 mRNA transcripts would, in cases of normal ploidy, contain equal ratios of each allele (1:1 ratio). If, however, an aneuploidy was present, then the ratio would shift to 2:1 (Fig. 1). Although small, this study is significant in that the analysis of ten cases with trisomy 21 demonstrated that fetal Down syndrome could be detected with a sensitivity of 90% and a specificity of 96%. This approach, then, appears to be very promising. The drawback of this approach, as currently used, is that it relies on the fetus being heterozygous for the SNP locus being interrogated. This constellation might be infrequent, particularly in certain ethnicities. Consequently, the number of SNP targets will need to be expanded and, if necessary, so will the number of chromosome-21-specific cff-RNA species.
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Figure 1 Schematic representation of the non-invasive detection of fetal Down syndrome via the analysis of placentally derived PLAC4 cf-RNA. cff-RNA, placentally derived cell-free fetal messenger RNA; mRNA, messenger RNA; MS, mass spectroscopy; RT-PCR, real-time polymerase chain reaction; SNP, single nucleotide polymorphism.
In a second study, Lo and colleagues attempted to overcome some of these issues by examining another technological breakthrough: digital PCR, a process whereby very small changes in template copy numbers can be accurately enumerated.17 The advantage of this technology over mass spectrometry is that it does not rely on the fetus being heterozygous for a particular SNP locus, i.e. measurements can be conducted if the fetus is also homozygous for the SNP locus in question. Not only would this greatly simplify the analysis, it would no longer be restricted to particular genetic constellations.
Trisomy 18: detectable via epigenetic modification of the maspin gene? Dennis Lo’s Hong Kong group was also instrumental in showing that epigenetically modified cff-DNA sequences might be suited for the detection of fetal aneuploidies. Epigenetic modifications are somatic alterations of the DNA that do not alter the genetic sequence but that do affect gene expression. A widely described form arises via the methylation of cytosine nucleotides at the so-called CpG islands. If a genetic sequence is heavily methylated, also termed hypermethylated, then it will tend not to be expressed. A classic example is the second female Xchromosome, where gene expression is largely shut down by epigenetic mechanisms. As epigenetic differences might exist between maternal and fetal cf-DNA, the Hong Kong group set out to identify genetic loci that differ in the methylation pattern between mother and fetus, the rationale being that it might be possible to exploit such differences and to distinguish
between fetal and maternal cf-DNA fragments that have the same nucleotide sequence. In this context, a clear different was noted between the fetal and maternal methylation status of the maspin gene: the promoter of the gene is not methylated in the placenta but is hypermethylated in maternal blood.18 These differences can be exploited by the use of a bisulfite-conversion step, whereby unmethylated cytosine is converted into uracil whereas the methylated cytosine is left unchanged, to generate fetal DNA sequences that can be readily discriminated from maternal ones, for instance by mass spectrometry (Fig. 2). In their initial investigations, Lo and colleagues were able to show that hypomethylated fetal maspin cff-DNA sequences could be detected and quantified reliably by realtime PCR, and that the behaviour of these molecules was very similar to more classic cff-DNA sequences, such as SRY. As the mapsin gene is located on chromosome 18, the group then set out to determine whether analysis of this locus could be used for the non-invasive detection of trisomy 18, for instance by a epigenetic allelic ration (EAR) approach.19 In this assay, two alleles are quantified relative to each other, again yielding a 1:1 ratio in cases with normal ploidy or a 1:2/2:1 ratio in aneuploid cases (Fig. 2). For their analysis, the group made use of a of a singlebase A/C variation in the promoter region of this gene. Although this approach is interesting, the authors indicate that it might be technically challenging, as they could not achieve a clear discrimination between euploid and aneuploid cases even when examining genomic DNA obtained from pertinent placentae. As such, it is not surprising that no clear assessment could be made regarding the analysis of maternal plasma samples, as only three samples were examined from trisomy 18 cases. An additional limit of this approach is that an absolute requirement is that the mother is homozygous C/C for the single-base A/C variation being investigated, as the presence of maternal A alleles will confound the assay. It is likely that the chief technical problem to be overcome is the aggressive nature of the bisulfite conversion, which can lead to an enormous destruction of input template. This event is crucial because of the very small numbers of cff-DNA sequences in a given maternal plasma sample, as any destruction of this valuable material will seriously hamper the subsequent analysis. Hence, a more optimal approach, which provides an effective conversion of unmethylated fetal cytosine sequences, will need to be sought. Alternatively, the focus could be placed on fetal sequences, which are hypermethylated whereas the corresponding maternal sequence is hypomethylated. This condition is met by the RASSF1A gene.20 Unfortunately, as this gene is located on chromosome 3, it will not prove to be useful for the detection of any of the most common fetal aneuploidies.
cff-DNA as a screening marker for fetal aneuploidies: a useful approach? One of the advantages of real-time PCR analyses of cff-DNA is that they offer a quantitative indication of the amount of fetal sequences present in a given sample.21 In this manner,
Recent progress in non-invasive prenatal diagnosis
Heterozygous maspin locus in placenta (unmethylated)
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Figure 2 Schematic representation of the non-invasive detection of fetal trisomy 18 via the analysis of epigenetically modified maspin sequences. cff-RNA, placentally derived cell-free fetal messenger RNA; MS, mass spectroscopy; PCR, polymerase chain reaction.
elevations in cff-DNA have been noted in pregnancies affected by preeclampsia, or those at risk of this disorder.22 In addition, discrete elevations have been noted in pregnancies with trisomy 21 fetuses, but not those with trisomy 18.23,24 These findings have suggested that cff-DNA could serve as a screening marker to detect pregnancies at risk for such fetal anomalies. Although this approach might be worth considering, it should be noted that cff-DNA levels can only be reliably quantified using multi-copy sequences on the Y chromosome, such as the DYS14 locus.25 As such, these analyses are restricted to cases in which the fetus is male. Although epigenetically modified genetic loci such as the RASSF1A gene could serve as fetal gender-independent markers, it is currently unclear how precise the determination of fetal cfDNA levels via the analysis of this sequence is. It should also be noted that these analyses are costly and so perhaps might be too expensive to be considered as a screening tool.
Discussion of future developments Although certain fetal genetic traits can now be determined non-invasively with great confidence, and although promising approaches appear to be on the horizon, a lot more work is required before the determination of complex Mendelian disorders or fetal aneuploidies becomes a clinical reality. The use of fetal cells enriched from maternal blood might, in future, be facilitated by the combined use of new markers and optimized microscopic platforms that permit
their automated detection. However, even if this were to become a reality, it would probably not be suitable for high-throughput analysis of large sample numbers; this feature would be possible via the analysis of cff-DNA or cff-RNA as all the required steps are highly amenable to automation. Several developments are necessary for this occur, including the use of mass spectrometric instruments such as the MassArray, marketed by Sequenom. This equipment needs to be optimized for a routine clinical environment, as certain steps (such as the extension and purification of PCR products that form the basis this analysis) are not suitable for rigorous day-to-day diagnostic use. Despite this premise, it is highly likely that this tool will become an important part of future cff-DNA analyses. In addition, the preparation of samples for cff-RNA or epigenetically modified fetal cf-DNA sequences needs to be improved and made more efficient to ensure that sufficient fetal template molecules are obtained for the subsequent PCR analysis. The highlight of recent studies is certainly cff-RNA, which indicates that this approach might be useful in determining fetal aneuploidies. However, it should be noted that this study was performed on a very small number of samples (10 cases and 56 controls) and, hence, these results need to be verified in a large multi-centre study, perhaps in a similar setting such as NIFTY or SAFE. Also still unclear is the number of mRNA transcripts and SNP loci that need to be targeted, and whether the use of digital PCRbased approaches will render these concerns superfluous.
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Practice points Invasive prenatal diagnosis is associated with increased risk of fetal loss. Non-invasive analysis of fetal genetic traits is already being used for fetal sex and RHD determination. Fetal Down syndrome could be detected with a sensitivity of 90% and a specificity of 96% by a non-invasive procedure via the analysis of cffRNA. Trisomy 18 might be detectable via the analysis of epigenetic modifications of the maspin gene, but the applicability is very limited. The quantitative use of cff-DNA levels is limited by the current need for high copy sequences such as DYS14.
Research agenda Increase the quality and quantity of fetal genetic materials in maternal circulation for analysis. Optimize approaches (such as mass spectrometric analysis) for sensitive, specific and quantitative detection of fetal loci, which can used for the determination of aneuploidies. Perform large multi-centre studies.
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8. Di NE, Ghezzi F, Vitucci A, et al. Prenatal diagnosis of betathalassaemia using fetal erythroblasts enriched from maternal blood by a novel gradient. Mol Hum Reprod 2000;6:571e4. 9. Garvin AM, Holzgreve W, Hahn S. Highly accurate analysis of heterozygous loci by single cell PCR. Nucleic Acids Res 1998; 26:3468e72. 10. Babochkina T, Mergenthaler S, De NG, et al. Numerous erythroblasts in maternal blood are impervious to fluorescent in situ hybridization analysis, a feature related to a dense compact nucleus with apoptotic character. Haematologica 2005;90: 740e5. 11. Prieto B, Candenas M, Ladenson JH, Alvarez FV. Comparison of different CD71 monoclonal antibodies for enrichment of fetal cells from maternal blood. Clin Chem Lab Med 2002;40: 126e31. 12. Kolialexi A, Vrettou C, Traeger-Synodinos J, et al. Noninvasive prenatal diagnosis of beta-thalassaemia using individual fetal erythroblasts isolated from maternal blood after enrichment. Prenat Diagn 2007;27:1228e32. 13. Coata G, Tilesi F, Fizzotti M, et al. Prenatal diagnosis of genetic abnormalities using fetal CD34þ stem cells in maternal circulation and evidence they do not affect diagnosis in later pregnancies. Stem Cells 2001;19:534e42. 14. Guetta E, Gutstein-Abo L, Barkai G. Trophoblasts isolated from the maternal circulation: in vitro expansion and potential application in non-invasive prenatal diagnosis. J Histochem Cytochem 2005;53:337e9. 15. Lo YM, Chiu RW. Prenatal diagnosis: progress through plasma nucleic acids. Nat Rev Genet 2007;8:71e7. 16. Lo YM, Tsui NB, Chiu RW, et al. Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection. Nat Med 2007;13:218e23. 17. Lo YM, Lun FM, Chan KC, et al. Digital PCR for the molecular detection of fetal chromosomal aneuploidy. Proc Natl Acad Sci U S A 2007;104:13116e21. 18. Chim SS, Tong YK, Chiu RW, et al. Detection of the placental epigenetic signature of the maspin gene in maternal plasma. Proc Natl Acad Sci U S A 2005;102:14753e8. 19. Tong YK, Ding C, Chiu RW, et al. Noninvasive prenatal detection of fetal trisomy 18 by epigenetic allelic ratio analysis in maternal plasma: theoretical and empirical considerations. Clin Chem 2006;52:2194e202. 20. Chan KC, Ding C, Gerovassili A, et al. Hypermethylated RASSF1A in maternal plasma: A universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis. Clin Chem 2006;52:2211e8. 21. Lo YM, Tein MS, Lau TK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62: 768e75. 22. Hahn S, Holzgreve W. Fetal cells and cell-free fetal DNA in maternal blood: new insights into pre-eclampsia. Hum Reprod Update 2002;8:501e8. 23. Lo YM, Lau TK, Zhang J, et al. Increased fetal DNA concentrations in the plasma of pregnant women carrying fetuses with trisomy 21. Clin Chem 1999;45:1747e51. 24. Zhong XY, Burk MR, Troeger C, Jackson LR, Holzgreve W, Hahn S. Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses. Prenat Diagn 2000;20:795e8. 25. Zimmermann B, El-Sheikhah A, Nicolaides K, Holzgreve W, Hahn S. Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma. Clin Chem 2005;51: 1598e604.