Cell-Free Fetal DNA in Maternal Blood: New Possibilities in Prenatal Diagnostics1)

DOI 10.1515/labmed-2012-0078 J Lab Med 2012; aop

Molecular-Genetic and Cytogenetic Diagnostics Editor: H.-G. Klein

Markus Stumm *, Rolf-Dieter Wegner and Wera Hofmann Cell-free fetal DNA in maternal blood: new possibilities in prenatal diagnostics1)

Abstract: Cell free fetal DNA (cff-DNA) in maternal blood of prenatal diagnostics, it has been necessary so far to has given rise to the possibility of new non-invasive obtain placental or fetal material through chorionic villus approaches in prenatal genetic diagnoses. In contrast to sampling (CVS) in the first trimester of pregnancy or by the established invasive techniques chorionic villi sam- amniocentesis (AC) in the second trimester of pregnancy. pling and amniocentesis, both associated with specific Both methods, however, are invasive and fraught with a risk (0.5 % – 1 % ) for procedure-related abortions, cff-DNA procedure-related miscarriage risk of 0.5 % – 1 % , so that is simply gained by maternal venous blood sampling, they are performed only after a thorough clinical indica- without any risk for the embryo or fetus. Therefore, cff- tion [1] . For the detection of the most common fetal aneu- DNA offers the possibility for riskless genetic diagnoses ploidies (trisomy 13, 18 and 21), non-invasive screening of ongoing pregnancies. Molecular genetic techniques are tests are used additionally that are based on ultrasound already used for the qualitative detection of specific fetal and the determination of hormone and protein para- sequences, such as paternal inherited or spontaneous meters in maternal serum. This is not a diagnostic pro- originated (de novo) mutations. Until recently, the intro- cedure, but merely risk assessments that due to limited duction of digital PCR and next generation sequencing sensitivity and specificity result in a relatively large technologies has shown that a reliable quantitative detec- number of false positive and false negative results (about tion of mutant alleles as well as of clinical relevant aneu- 5 % ). These upset pregnant women and lead ultimately to ploidies (Trisomy 13, 18 and 21) from fetal DNA in maternal invasive procedures that are not really necessary [2] . The blood is also possible. great hope of prenatal diagnosticians therefore is to be able to use safe non-invasive diagnostic procedures that Keywords: aneuploidy; cell free fetal DNA in maternal do not entail additional risk for an existing pregnancy. blood; non-invasive prenatal diagnosis. After the first not-so-successful diagnostic tests on fetal cells from maternal blood [3] , the discovery of fragmented cell-free fetal DNA in maternal plasma [4] led to promising 1) Original German online version at: http://www.degruyter. new approaches in non-invasive prenatal diagnostics, and com/view/j/labm.2012.36.issue-5/labmed-2011-0030/ labmed-2011-0030.xml. The German article was translated by now in several European countries and in the U.S. special Compuscript Ltd. and authorized by the authors. non-invasive genetic diagnostic methods on fetal DNA *Correspondence: PD Dr. rer. nat. Markus Stumm, Zentrum from maternal blood are already part of prenatal diagnos- f ü r Pr ä nataldiagnostik und Humangenetik Kudamm 199, tic pregnancy care. A brief chronology of the development Kurf ü rstendamm 199, 10719 Berlin, Germany, is shown in Table 1. Tel.: + 49 30-88043150, Fax: +49 30-88043176, E-Mail: [email protected] Markus Stumm and Rolf-Dieter Wegner: Center for Prenatal Diagnostics and Human Genetics Kudamm 199 , Berlin , Germany ; and BG Berlin-Genetics GmbH, Berlin , Germany Fetal DNA in maternal blood Wera Hofmann: LifeCodexx AG, Constance , Germany The discovery that fragmented cell-free fetal DNA (cff- DNA) occurs in maternal plasma and serum [4] opened up entirely new possibilities for non-invasive prenatal diagnostics. This cff-DNA originates primarily from tropho- Introduction blast cells and passes through the fetal-maternal transac- tion in the chorion to the maternal circulation [27] . It is Prenatal diagnostics is an integral part of gynecologi- detectable as early as from the fifth week of pregnancy cal practice. To perform a genetic test in the context and persists to the end of the pregnancy [28] . It occurs in 2 Stumm et al.: Cell-free fetal DNA in prenatal diagnostics

Year Event Method Reference

1997 Discovery of cell-free fetal DNA in the maternal blood, and sex determination Polymerase chain reaction (PCR) [4] 1998 Determining the RhD blood groups PCR [5] 2000 Diagnostics of a dominant mutation (myotonic dystrophy) PCR [6] 2001 Sex verification and RhD typing commercially available PCR [7] 2002 Detection or exclusion of recessive mutations (congenital adrenal hyperplasia PCR [8, 9] and cystic fibrosis) 2002 Detection of paternally inherited HLA haplotypes Real time PCR (RT-PCR) [10] 2004 Detection of fetal polymorphisms through parental haplotype analyses PCR and mass spectrometry [11] 2007 RhC, RhE and Kell blood group typing PCR [12] 2008 Detecting trisomy 13, 18 and 21 Next generation sequencing (NGS) [13, 14] 2010 Detection of fetal whole genome through parental haplotype analyses NGS [15] 2011 Four large studies show a high sensitivity and specificity in the detection of NGS [16 – 19] trisomy 21 2011 Trisomy 21 test commercially available in the U.S. NGS [20] 2011 Proof of familial microdeletion NGS [21] 2012 High specificity and sensitivity in the detection of trisomy 13 and 18 NGS [22] 2012 Establishment of a non-invasive paternity test NGS [23] 2012 Evidence of 22q11.2 microdeletion NGS [24] 2012 Detection of additional autosomal and gonosomal aneuploidies NGS [25] 2012 Trisomy 21 test commercially available also in Europe NGS [26]

Table 1 Chronology of non-invasive genetic diagnostics of cell-free fetal DNA from maternal blood. all pregnancies [29] , but due to its short half-life (approx. of disease if the mother is the carrier of a relevant muta- 16 min), it cannot be detected anymore only a few hours tion. This putative risk can then be clarified further by an after birth [30] . Thus, there is no false diagnosis because invasive procedure using CVS or AC. However, if there is of persistent DNA fragments of a previous pregnancy. In no proof of Y-specific sequences in the mother ’ s blood, it the maternal circulation, the cff-DNA is present together must be assumed that the present pregnancy involves a with maternal DNA fragments; in maternal plasma, it female fetus that then does not carry a risk of severe clini- accounts on average for about 5 % – 10 % , but this can vary cal manifestation in the case of X-chromosomal recessive depending on the week of pregnancy. Performing a correct heredities. In such cases, it is then possible to dispense genetic diagnosis on fetal DNA with such a high maternal with invasive genetic diagnostics. Sex determination using DNA background is the real challenge. The first studies cff-DNA from maternal blood succeeds with a certainty on fetal DNA therefore focused on qualitative parameters, of more than 95% and has already been used in clinical i.e., genetic sequences of the fetus not contained in the diagnostics [31, 32]. As a further clinical application, non- maternal genome, such as Y-DNA sequences of male invasive sex determination was used in the treatment of fetuses or paternally inherited alleles and de novo muta- congenital adrenal hyperplasia. Here female fetuses can tions (see Tables 1 and 2). be specifically treated with corticosteroids to prevent virilization [33] . As a further option, a non-invasive sex determination provides additional information in unclear The qualitative detection of specific ultrasound findings with conspicuous fetal genitalia. fetal sequences in maternal blood Rhesus-D genotyping Proof of Y-chromosomal sequences Rhesus D (RhD) positive fetuses from RhD-negative If one can detect Y-specific sequences in maternal blood, mothers have an increased risk, already during preg- it can be assumed that the present pregnancy involves a nancy, of iso-immunization, hemolytic diseases and mis- male fetus. A medical indication of prenatal sex deter- carriage. But through the prophylactic use of anti-RhD mination is given in connection with families with sex- immunoglobulin, these risks can be prevented. If it is linked genetic diseases, such as hemophilia or Duchenne of clinical significance, the fetal RhD status can also be muscular dystrophy (DMD). Here the fetus has a 50 % risk determined prenatally. This can be done by means of fetal Stumm et al.: Cell-free fetal DNA in prenatal diagnostics 3

genotyping from chorionic villi or amniotic fluid cells, but before the invasive procedure this requires an appropriate allo-immunization of the mother. Without corresponding problems, the fetal RhD genotype can also be determined non-invasively from the cff-DNA from maternal blood [5, 34]. Through the 95 % 95 % 99 % in high-risk groups ∼ cases Individual Up to 100 % Up to 100 Individual cases Individual > > qualitative detection by PCR electrophoresis protocols or real-time PCR technologies, it is possible, similar to the non-invasive sex determination, to determine the presence of RhD gene sequences of an RhD-positive fetus in the plasma of an RhD negative woman [34, 35]. A meta- analysis showed that non-invasive fetal DNA tests have a

hemophilia cases Individual 95% sensitivity and can be performed starting from the eighth week of pregnancy [36] . After the 16th week of pregnancy, a sensitivity of actually 99 % can be achieved [37] . Due to the high sensitivity and specificity of these non-invasive RhD tests, appropriate methods were integrated at different prenatal centers early in routine diagnostics [31, 38, 39] . In some European countries, the non- invasive RhD diagnostic tests of RhD negative women are routinely offered [40] . An anti-RhD immunoglobulin Familial microdeletion, deletion 22q11.2Familial cases Individual Trisomy 13 13 Trisomy 18 Trisomy Trisomy 13 Trisomy Congenital adrenal hyperplasia hyperplasia adrenal Congenital fibrosis Cystic Hemoglobinopathies Myotonic dystrophy dystrophy Myotonic Achondroplasia Hemoglobinopathies HB Leopore disease Huntington ’ s disease injection then takes place only in the diagnosis of an RhD positive fetus. Non-invasive fetal DNA tests were also tested for the typing of other fetal-maternal incompatibility, such as RhC, RhE and Kell [12, 41], and are now also available as a diagnostic test for high-risk pregnancies [42].

The detection of monogenic diseases

The prenatal detection of monogenic changes is particu-

PCRlarly of clinical hemoglobinopathies Thalassemias, significance for diseases with a high popu- generation sequencing generation 21 Trisomy

xt generation sequencing generation xt Thalassemialation-specific prevalence. Examples case Individual include thalassemia Allele-specific PCR PCR Allele-specific Electrophoresis RT-PCR Allele-specific Method RT-PCR Allele-specific Diagnosticsand cystic fibrosis. Sensitivity Furthermore, the prenatal diagnosis of rare monogenic disorders also plays an important role in families with a previous history. Preliminary results from a non-invasive diagnosis of monogenic diseases with cff-DNA focused on the detection or exclusion of paternally inherited autosomal-dominant mutations in maternal plasma. In 2000, the first successful non-invasive diagnosis of cff-DNA was made for the detection or exclusion of a paternally inherited mutation that causes myotonic dystrophy [6] . Other publications describe the successful use of non-invasive cff-DNA diagnostics in connection with achondroplasia [43] , hemoglobinopathies [44] and Huntington ’ s disease [45] . Further- more, cff-DNA was successfully tested in the HLA typing

Examples of qualitative and quantitative parametric analyses. analyses. parametric quantitative and qualitative of Examples as part of the HLA-matching for planning hematopoietic stem cell transplantation for sick siblings [10] and in non- excluding the paternal mutation excluding the paternal Microdeletion diagnostics sequencing generation Next HLA typing and parentage testing parentage HLA typing and genome whole fetal Haplotyping of analyses Qualitative dosage diagnostics using relative mutation Genetic Aneuploidy diagnostics diagnosticsAneuploidy Digital RT-PCR Allele-specific Ne Next test paternity HLA matching, sequencing Targeted 21 Trisomy cases Individual Diagnostics of autosomal-recessive diseases by by diseases autosomal-recessive Diagnostics of Blood group typingBlood diseases autosomal-dominant Diagnostics of electrophoresis PCR with specific Allele PCR Real-time RhD Table 2 Table Qualitative analyses Qualitative Sex determination Real-time PCRinvasive paternity SRY diagnostics [23] . 4 Stumm et al.: Cell-free fetal DNA in prenatal diagnostics

The non-invasive detection of autosomal-recessive The quantitative detection of fetal or maternally inherited autosomal-dominant disease proved much more difficult, because the sole evidence of sequences in maternal blood a mutant allele does not provide any information on the genotype of the fetus. A methodical procedure for the detection of single mol- In autosomal-recessive diseases, via the exclusion of ecules allows not only the quantitative analysis of specific a specific paternally inherited mutation, a disease of the alleles, but also, of course, for the determination of the fetus can be ruled out. Because if the fetus has received chromosomal dose and thus opens the door for non-inva- the normal allele of the father, no disease can occur, no sive diagnostics of fetal aneuploidy (see Table 2). matter whether the fetus has inherited the mutated or Aneuploidies, from the time of conception, are the wild-type allele of the mother. Only a possible carrier leading cause of death in people. The most common aneu- status of the maternally inherited mutation cannot be ploidy among newborns is trisomy 21 with an incidence of ruled out. Based on the exclusion of paternally inherited 1:500 to 1:700. The risk of pregnancy with aneuploidy is mutations, the first non-invasive diagnostic tests were directly correlated with the mother’ s age. While mothers carried out on cff-DNA for congenital adrenal hyperpla- at the age of 20 years have an aneuploidy risk in the range sia (CAH) [9] , cystic fibrosis (CF) [8] and thalassemia [46] . of 0.2 % , this risk is increased in women aged 35 % to 0.5 % This method is not applicable if the father and mother and 5 % in women aged 45. carry the same mutation. To have a diagnostic agent Through the change in lifestyle and education struc- available even for such situations, the working group of tures, the industrialized countries have seen in recent Chiu and Lo developed the RMD method. RMD stands for decades a shift in the pregnancy age from the second Relative Mutation Dosage: it measures and compares the to the third decade of life. A study of the English and relative content of the mutant and the normal allele of a Welsh Down syndrome registry for the period 1989 – 2008 specific gene quantitatively [47] . The RMD is based on the described an increase in diagnosed trisomy 21 cases by method of digital PCR, in which hundreds or thousands 71% , which is mainly attributable to the increase in mater- of amplified DNA molecules are diluted to single-mole- nal age. More than 20 % of pregnancies occur today namely cule concentrations and then counted. By comparison of in women who are older than 35 years [49] . The demo- the relative number of the individual molecules, a quan- graphic shift to increased maternal age and the associated titative analysis of mutant and non-mutant alleles in increased risk of aneuploidy thus require also a shift in maternal plasma is possible. If a woman is a heterozygous prenatal diagnostics, and reliable non-invasive diagnos- carrier of a recessive mutation and the fetus carries the tic methods could make an important contribution. The same mutation in the heterozygous state, the proportion methods developed in recent years for single molecule of mutated and non-mutated sequences is equal. If the detection, digital PCR and especially the next-generation fetus is a homozygous carrier of this mutation, however, sequencing (NGS = Next generation sequencing) play a disproportionately more mutated than non-mutated crucial role [50] . Because digital PCR and Massive Para- sequences are found in maternal plasma. Accordingly, llel Sequencing can reliably measure the increase of fetal a homozygously normal fetus exhibits proportionally DNA in maternal plasma, which is caused by the gain of a more non-mutated sequences in maternal plasma. With aneuploid chromosome. the development of the RMD method it is also possible For determining the relative chromosomal dosage to diagnose a maternally inherited autosomal-dominant using digital PCR, the proportion of chromosome 21 mole- mutation non-invasively. By way of RMD, it was possi- cules in maternal plasma was compared with that of a ble to diagnose the transmission of maternal mutations reference chromosome. The ratio of the proportion of in thalassemia, hemophilia and hemoglobinopathies chromosome 21 DNA in a fetal trisomy 21 and the propor- [47, 48] . tion of chromosome 21 DNA of a reference patient collec- In 2010, the working group of Lo succeeded for the tive without fetal trisomy 21 changes or increases because first time in showing the entire genome of a fetus from the there are correspondingly more chromosome 21 specific cff-DNA from maternal blood and performing thalassemia sequences in maternal plasma. The quantitative sensiti- diagnostics with the help of parental haplotypes [15]. vity increases with the proportion of fetal DNA in mater- In summary, this leads to the realization that by using nal blood, and with the number of digital PCR analyses the new technologies, any monogenic disease can be performed. For the accurate detection of trisomy 21 with diagnosed by means of non-invasive cff-DNA analysis in a 25% fetal DNA proportion in maternal plasma, 8000 future. digital PCR reactions are required, which will enable the Stumm et al.: Cell-free fetal DNA in prenatal diagnostics 5 use of this method in the clinical setting only by means of quantified. In the case of fetal trisomy 21, in comparison fully automated platforms [51] . The clinical establishment to other chromosomes, slightly more chromosome 21 spe- of non-invasive trisomy 21 diagnostics using NGS has been cific sequences are detected. This approach also provides a reality in the U.S. since October 2011 [20] and will also a diagnostic tool to test not only for a specific example of arrive on the European market in 2012 [26] . trisomy, such as trisomy 21, but it also has the theoretical NGS technologies produce, in comparison to Sanger potential of a DNA analysis of the entire genome, wherein sequencing, much larger amounts of data at signifi- aneuploidies and mutations can be detected. cantly lower costs. In the Sanger sequencing, per day and That this is not quite as simple was already demon- automated sequencer (e.g., ABI 3730xl) a maximum of strated in the study by Fan et al. [14] , which was able by around 40 megabases are sequenced. An NGS sequencer means of MPS to detect trisomy 13 and trisomy 18, but with (e.g., HiSeq2000, Illumina, San Diego, USA) manages a lower selectivity compared to trisomy 21. These differ- 55 gigabases in the same period. But the high sequence ences are mainly due to the different GC content of the yield also comes at a price, because the accuracy of the individual chromosomes. This demonstrated that a quan- sequencing ( = base calling) is clearly inferior in NGS titative MPS analysis of chromosomes with high or low GC method compared to the Sanger method. Therefore, the content was much more difficult and that a reliable non- NGS methods are also mainly used for resequencing. In invasive diagnosis required the development of sequenc- resequencing, NGS reads are compared with a reference ing protocols and bioinformatic analysis algorithms. genome to find the places to which the NGS reads corre- Other studies have shown, in the meantime, that the spond, so that a kind of genomic map of matching reads is use of specific enrichment methods and the modification created. Through a chromosomal assignment and propor- of bioinformatic calculations can allow for the reliable tional quantitative analysis, information can thus be pro- detection of trisomy 13 and 18 [22, 51, 53] , while also detect- vided on the gain or loss of genomic sequences, thereby ing other autosomal and gonosomal aneuploidies [25] . allowing for aneuploidy diagnostics by NGS. In 2008, the groups of Dennis Lo and Stephen Quake described for the first time the use of Massive Parallel Clinical use of non-invasive cff-DNA Sequencing (MPS) for the non-invasive detection of fetal aneuploidies in cff-DNA [13, 14] . Both Chiu and Fan showed diagnostics in their independent cohort studies that trisomy 21 from cff-DNA from maternal blood can be detected with 100 % Since the discovery of fetal DNA in maternal blood in 1997, sensitivity and specificity. We were able to confirm, in our because of many research projects much progress has own proof of principle study, the reliability of the non- been made, and cff-DNA is already being used in clinical invasive diagnosis of trisomy 21 on two different NGS plat- diagnostics. The non-invasive analysis of the fetal sex and forms [51] . The first results were promising, and have in the RhD status from cff-DNA is in some European countries meantime been confirmed in several large clinical studies already firmly established in prenatal diagnostics. The [16 – 19] . Summarizing the results of these studies, the diag- corresponding laboratory protocols were revised con- nostic sensitivity of the non-invasive trisomy 21 test in high- stantly. Through the integration of universal fetal markers risk patients was 99.1% with a specificity of 99.7% [52] , and (e.g., RASSF1A) as internal experimental controls, the sen- thus provides better results than the non-invasive screening sitivity and specificity was improved significantly and is method routinely used in clinical practice that achieve a now in the range of 99.6 % to 99.9 % [54] . maximum sensitivity and specificity of approximately 95 % . Since October 2011, a test method for the non-invasive First results from our own prospective, blinded, multi-center detection of trisomy 21 has been available commercially study also showed excellent clinical accuracy for the trial in the U.S. through the company Sequenom [Sequenom, design of the new commercial PraenaTest by the company San Diego, USA]. A similar method is to be available on the LifeCodexx (LifeCodexx, Constance, Germany) [26] . European market later this year, offered by the company In contrast to digital PCR, due to the selection of some LifeCodexx. This, of course, raises the question about the selected loci based only on the analysis of less different possibilities and limitations of non-invasive methods. DNA fragments, MPS uses several million DNA fragments Are they suited to fully take the place of the conventional from a single sample for analysis. The corresponding frag- non-invasive aneuploidy screening and/or invasive proce- ments are sequenced simultaneously in short segments, dures, or should the new non-invasive molecular genetic sorted by complex bioinformatic analyses and information methods be integrated meaningfully into the existing from a genomic database by chromosomal origin and then methods of prenatal diagnostics ? 6 Stumm et al.: Cell-free fetal DNA in prenatal diagnostics

At the present time, this question has not been clearly In our assessment at this time, the aneuploidy diag- answered, because the diagnosis of aneuploidy using nosis from cff-DNA in the context of the first trimester cff-DNA achieves in large studies of trisomy 21 “ only ” a screening (ETS) makes the most sense. Especially in sensitivity of about 99.7 % and a specificity of 99.7 % [52] , women who in the context of conventional screening and if trisomy 13 and 18 are included, a sensitivity of 99 % exhibit an increased trisomy 21 risk after NT measurement and a specificity of 98% [22] . Is this enough sensitivity and and serum analysis, the test can be offered as an interme- specificity for a diagnostic test, or must a diagnostic test diate step. If the non-invasive molecular genetic aneu- achieve a sensitivity and specificity of 100% ? It should ploidy test is unremarkable, a direct invasive test can be be noted that cff-DNA originates mainly from trophoblast avoided. An unremarkable ultrasound at the beginning cells and therefore can only reach the diagnostic accu- of the 2nd trimester would then bring additional relief. racy of a direct preparation of chorionic villus sampling In case of a conspicuous ultrasound, the patient could (CVS). Chromosomal mosaics or fetoplacental discrepan- then still be offered an amniocentesis. Detailed genetic cies occur in invasive cytogenetic CVS with a frequency of counseling according to the requirements of the Genetic 1 % – 2 % in [1] , so that mathematically 100 % test sensitivity Diagnostics Act [55] is absolutely necessary in this and specificity cannot be achieved. These figures must, context. Overall, a responsible use of the new methods of course, be factored in when assessing the reliability of could result in only a fraction of patients with a false non-invasive aneuploidy diagnostics, as well as in con- positive ETS undergoing invasive diagnostics, and then nection with CVS. This means that a conspicuous finding miscarriages caused by invasive diagnostics would also is not always representative for the fetus and possibly be reduced significantly. subject to further diagnosis. A limiting factor at the moment is the high price of Further diagnosis would be necessary also with a con- non-invasive testing. Recent technological developments, spicuous finding, since the non-invasive DNA tests, similar such as even higher sequencing performance, strategies to interphase fluorescence hybridization or QF-PCR, for enrichment of cff-DNA and/or the selective detection cannot differentiate between a free and a translocation of specific sequences, could lead to a significant cost trisomy. That is, in the case of non-invasively detected reduction of NGS technologies. In addition, technological aneuploidy (e.g., trisomy 13 and 21) not confirmed by advancements will also increase test efficiency, which will invasive cytogenetic diagnostics, a chromosome analysis lead to smaller chromosomal imbalances being detected of both parents would be indicated in order to exclude the reliably on a genome-wide level. Overall, NGS technolo- possible increased risk of recurrence of a familial translo- gies currently hold the greatest potential as non-invasive cation (e.g., Robertsonian translocation). cff-DNA tests in aneuploidy diagnostics. But as long as In addition, it should be noted that previous studies larger-scale studies have not shown the successful appli- were all carried out on populations with high-risk preg- cation in low-risk patient populations, the established nancies. For example, whether the fetal DNA concen- structures should not be abandoned, and the non-invasive tration and hence the diagnostic reliability in other risk tests should be offered only to patients with an increased groups is correspondingly high cannot currently be aneuploidy risk after ETS. answered. From a cytogenetic perspective, the currently usable Conflict of interest statement non-invasive testing methods provide only a restricted view of the fetal karyotype, since only trisomy 21 or, at the Authors ’ conflict of interest disclosure: The authors most, trisomy 13 and 18 are detected. In a pure trisomy 21 stated that there are no conflicts of interest regarding the test only 37% of all chromosomal abnormalities would publication of this article. thus be detected in our own patient population. Research funding: None declared. From a clinical perspective, the first-trimester screen- Employment or leadership: PD Dr. Stumm and Prof. Dr. ing also detects additional important parameters and not Wegner are member of the Center for Prenatal Diagno- only the risk aneuploidy. For both the ultrasound screen- stics and Human Genetics Kudamm-199. PD Dr. Stumm ing and the biochemical markers can capture other fetal and Prof. Dr. Wegner are shareholders of BG Berlin- abnormalities and pregnancy complications. This infor- Genetics GmbH. Dr. Hofmann is employee of Lifecodexx mation does not result from non-invasive diagnostics from AG. cff-DNA either. Honorarium: None declared. Stumm et al.: Cell-free fetal DNA in prenatal diagnostics 7

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