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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 1) possibilities in prenatal diagnostics 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 diag- nostics. 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 para meters, of more than 95 % and has already been used in clini cal 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 pres- ence 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 inte- grated at different prenatal centers early in routine diag- nostics [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.

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