WO2005021793A1 - Prenatal diagnosis of down syndrome by detection of fetal rna markers in maternal blood - Google Patents

Abstract

The present invention relates to method for prenatal diagnosis of Down's syndrome-affected pregnancies. In the method fetal RNA, in particular placentally derived RNA indicative of Down's syndrome is detected in a maternal blood sample. Preferably in the method the placental RNA is detected in maternal blood plasma or serum obtained in the first trimester of pregnancy using RT-PCR for detection and preferably quantification of the RNA.

Description

PRENATAL DIAGNOSIS OF DOWN SYNDROME BY DETECTION OF FETAL RNA MARKERS IN MATERNAL BLOOD

Field of the invention The present invention relates to non-invasive methods for prenatal diagnosis of a Down's syndrome-affected pregnancy by detection of fetal, in particular placental RNA, the presence or quantity of which is indicative of Down's syndrome, in maternal blood samples taken from a pregnant subject.

Background of the invention Trisomy 21, commonly referred to as Down's syndrome, is the most frequent chromosome disorder seen in newborn infants and is the leading genetic cause of moderate mental retardation. Since the early 1970's, testing for this disorder became feasible with the advent of amniocentesis. Unfortunately, amniocentesis, chorion villus sampling and other invasive methods for sampling fetal cells for chromosomal, genetic or biochemical analysis are associated with a recognized fetal loss rate of at least 1% and therefore only offered to women at increased risk for aneuploidy. For these reasons non-invasive methods for prenatal diagnosis of Down's syndrome-affected pregnancies have been developed, including measurement of nuchal translucency by ultrasound and maternal serum screening for markers such as human chorionic gonadofrophin (hCG), pregnancy-associated plasma protein- A, unconjugated estriol, inhibin A and the free β- subunit of hCG for first trimester screening. However, the false-positive rate of these non-invasive methods, even when used in combination, is such that for a definitive diagnosis most patients still will undergo amniocentesis with the associated risk of miscarriage. Recent interest in cell-free DNA in plasma and serum has led to the discovery of fetal DNA in maternal plasma (Lo et al., 1997, Lancet 350: 485-487; WO 98/39474). Despite the promising clinical use of fetal DNA in maternal plasma for non-invasive prenatal diagnosis, a number of challenges remain. E.g. for the analysis of abnormalities involving the quantitation of fetal DNA in maternal plasma, the Y- chromosome is commonly used as fetal-specific marker in women carrying male fetuses. This, however, limits the application of this technology to only the 50% of pregnancies carrying male fetuses. More recently, it was found that in addition to DNA, RNA is also present in the plasma of human subjects with neoplastic conditions (WO97/35589), as well as in non- neoplastic conditions (WO 03/009806). Also the presence of fetal RNA in the plasma of pregnant women has been established (Poon et al., 2000, Clin. Chem. 46: 1832- 1834). It has since been demonstrated that mRNA of placental origin can be easily detected in the maternal plasma (Ng et al., 2003, Proc. Natl. Acad. Sci. USA 100:

4748-4753; WO 02/33120). The surprising stability of fetal RNA in maternal plasma appears due to the fact that the fetal RNA circulates in a protected form, which seems to involve microparticles, which are predominantly placental in origin (Tsui et al., 2002, Clin. Chem. 48: 1647-1653; Ng et al., 2002, Clin. Chem. 48: 1212-1217;

Oudejans et al., 2003, Prenatal Diagnosis 23: 111-116). The presence of circulating placental RNA in maternal blood might be exploited for the development of a non-invasive diagnostic test for Down's syndrome-affected pregnancies. The development of such a test would, however, require the availability of placentally expressed RNA's, the presence or quantity of which would be indicative of a Down's syndrome pregnancy. Furthermore such RNA's should be detectable in maternal plasma, preferably already during early pregnancy, i.e. in the first trimester. Thus far such placental RNA's have not been characterized. Thus there is an as yet unmet need for the identification of placentally expressed RNA's that are predictive for Down's syndrome pregnancy and could be developed as new markers for Down's syndrome screening using detection of placental RNA in maternal plasma, preferably in the first trimester of pregnancy.

Description of the invention In a first aspect the present invention relates to a method for performing a prenatal diagnosis of a Down's syndrome-affected pregnancy. The method is based on detecting or inferring the presence of fetal RNA indicative of Down's syndrome in a maternal blood sample. The method comprises (a) detecting ex vivo a fetal RNA in a maternal blood sample; and, preferably (b) providing the diagnosis based on at least one of the presence, quantity or concentration of the fetal RNA. Step (a) of the method will usually involve (1) the extraction of fetal-derived or associated RNA from maternal blood, preferably from blood plasma or serum; (2) the application of some form of a nucleic acid amplification assay to the extracted RNA, whereby the extracted RNA may first be reverse transcribed to cDNA prior to amplification of the cDNA; and (3) the detection of one or more specific fetal RNA's or their amplification products. The amplification and detection steps (2) and (3) may be performed so as to allow either qualitative or quantitative detection of the fetal-derived RNA, depending upon the ultimate clinical relevance of the fetal RNA in question with respect to establishing the diagnosis of a pregnancy or fetus with Down's syndrome, as described herein. Such a fetal-derived RNA that may be used for diagnosing a Down's syndrome-affected pregnancy is herein referred to as a fetal marker RNA. If, for diagnostic purposes, the quantity of such a fetal marker in plasma needs to be corrected for biological and experimental variability, preferably the quantity of the fetal marker RNA is compared to the quantity of a second fetal RNA, determined under identical conditions. The second fetal RNA preferably is expressed from a different chromosome than chromosome 21 and is herein referred to as a fetal reference RNA. The method is preferably performed ex vivo on a blood sample that is obtained from a pregnant female. Either "fresh" blood plasma or serum, or frozen (stored) and subsequently thawed plasma or serum may be used for purposes of this invention. Frozen (stored) plasma or serum should optimally be maintained at storage conditions of -20 to -70 degrees centigrade until thawed and used. "Fresh" plasma or serum should be refrigerated or maintained on ice until used, with RNA extraction being performed as soon as possible. Blood may be drawn by standard methods into a collection tube, preferably siliconized glass, either without anticoagulant for preparation of serum, or with EDTA, sodium citrate, heparin, or similar anticoagulants for preparation of plasma. The preferred method if preparing plasma or serum for storage, although not an absolute requirement, is that plasma or serum be first fractionated from whole blood prior to being frozen. This reduces the burden of extraneous intracellular RNA released from lysis of frozen and thawed cells which might reduce the sensitivity of the amplification assay or interfere with the amplification assay through release of inhibitors to PCR such as porphyrins and hematin. Thus, in a preferred method of the invention, all nucleated and anucleated cell populations are removed from the blood sample prior to detection of fetal RNA. More preferably, the fetal RNA is detected in maternal blood plasma or serum. "Fresh" plasma or serum may be fractionated from whole blood by centrifugation, using gentle centrifugation at 300-800 x g for five to ten minutes, or fractionated by other standard methods. Particularly preferred in the fractionation of plasma or serum from whole blood is the addition of a second centrifugation step for five to ten minutes at about 20.000 to 30.000 x g, more preferably at about 25.000 x g to improve the signal to noise ratio in subsequent RNA detection methods. Since heparin may interfere with RT-PCR, use of heparinized blood may require pretreatment with heparinase, followed by removal of calcium prior to reverse transcription, as described (Imai et al., 1992, J. Virol. Methods 36: 181-184). Thus, EDTA is the preferred anticoagulant for blood specimens in which PCR amplification is planned. In the methods of the invention, the fetal marker RNA is usually detected in equal or less than 2 ml maternal blood, plasma or serum, more preferably in equal or less than 1.6, 0.8, 0.4, 0.2 or 0.1 ml of maternal blood, plasma or serum. In the methods of the present invention, the fetal RNA may be extracted from maternal body fluids, preferably whole blood, and more preferably plasma or serum using e.g. RNA extraction methods such as, but not limited to, gelatin extraction method; silica, glass bead, or diatom extraction method; guanidinium thiocyanate acid- phenol based extraction methods; guanidinium thiocyanate acid based extraction methods; guanidine-hydrochloride based extraction methods; methods using centrifugation through cesium chloride or similar gradients; phenol-chloroform based extraction methods; and/or other available RNA extraction methods, as are known in the art for use in extraction of intracellular RNA, including commercially available RNA extraction methods, e.g. by using or adapting or modifying the methods of Boom et al. (1990, J. Clin. Microbiol. 28: 495-503); Cheung et al. (1994, J. Clin. Microbiol. 32: 2593-2597); Boom et al. (1991, J. Clin. Microbiol. 29: 1804-1811); Chomczynski and Sacchi (1987, Analytical Biochem. 162:156-159); Chomczynski, (1993, Biotech. 15: 532-537); Chomczynski and Mackey (1995, Biotechniques 19: 942-945); Chomczynski and Mackey (1995, Anal. Biochem. 225: 163-164); Chirgwin et al. (1979, Biochem. 18: 5294-5299); Fournie et al. (1986 Anal. Biochem.158: 250-256); and WO97/35589. Particularly preferred RNA extraction methods for use in the methods of the invention are commercially available extraction methods suitable for extraction of intracellular, extracellular and in particular viral RNA, including e.g., TRIzol.TM. (Life Technologies); Trisolv.TM. (BioTecx Laboratories); ISOGEN.TM. (Nippon Gene); RNA StatTM. (Tel-test); TRI Reagent.TM. (Sigma); SV Total RNA Isolation System (Promega); RNeasy Mini Kit, QIAamp MinElute Virus Spin or QIAamp MinELute Virus Vacuum Systems (Qiagen, Hilden, Germany); Perfect RNA: Total RNA Isolation Kit (Five Prime-Three Prime Inc., Boulder, Colo.); or similar commercially available kit, wherein extraction of RNA may be performed according to manufacturer's directions, adapted to the maternal blood, serum or plasma. Most preferably the QIAamp MinELute Virus Vacuum System is used as it reduces the presence of aspecific bands in RT-PCR (see Figure 3). In a preferred embodiment, RNA is extracted from maternal blood, serum or plasma using a probe or probes that specifically hybridize to specific RNA species, such as but not limited to probes attached to solid substrates or probes attached to magnetic beads or particles, or probes wherein upon hybridization to a nucleic acid, an electrical gradient or magnetic gradient or density gradient can thereby enable extraction and/or separation of specific RNA species from the remainder of bodily fluid. Further, the RNA or cDNA derived therefrom may be hybridized to a solid substrate at a bio-electrical interface whereupon hybridization of a specific RNA, or cDNA derived therefrom, generates an electrical signal which may further be amplified and detected. Circulating extracellular DNA, including fetal-derived or associated extracellular DNA, is also present in maternal plasma and serum (see e.g. WO 98/39474). Since this DNA will additionally be extracted to varying degrees during the RNA extraction methods described above, it may be desirable or necessary to further purify the fetal RNA extract and remove trace DNA prior to amplification and/or detection of the fetal RNA. This may be accomplished using e.g. DNase in a method as described by Rashtchian (1994, PCR Methods Applic. 4: S83-S91). Following the extraction of the fetal RNA from a maternal blood sample one or more specific fetal marker or reference RNA's are detected therein in a qualitative and or quantitative manner so to allow the diagnosis of a pregnancy or fetus with Down's syndrome. Prior to detection, the specific fetal RNA's may be subjected to some form of a nucleic acid amplification assay to the extracted RNA, whereby the extracted RNA may first be reverse transcribed to cDNA prior to amplification of the cDNA. A fetal marker RNA for use in the methods of the present invention is an RNA that is expressed in the fetus, i.e. that is of fetal origin or fetally-derived. Preferably the fetal marker RNA is an RNA transcribed from a gene on chromosome 21. More preferably, the fetal marker RNA is an RNA transcribed from a gene present within or near the Down's syndrome critical region on chromosome 21. Such fetal marker RNA's are preferably detectable in a maternal blood sample during early pregnancy, more preferably in the first trimester of pregnancy and most preferably prior to week 17, 16, 15, 14, 13, 12, 11, 10, 9 or 8 of gestation. In order to facilitate detection, the fetal marker RNA further preferably is an RNA that is expressed in the placenta. More preferably, the placental RNA is an RNA that is of trophoblastic, in particular of extravillus origin, i.e. at least expressed in those types of placental cells. RNA's suitable as fetal marker RNA's include RNA's expressed from a gene of human chromosome 21 selected from the group consisting of TPTE (Accession No.: NM_013315), BAGE (Accession No.: NM_001187), LOC339635 (Accession No.: XM_293210), EIF3S5P (Accession No.: AP0030900), C21ORF99 (Accession No.: NM_153773), LOC343717 (Accession No.: XM_293177), LOC284812(Accession No.: XM_208251), LOC284813 (Accession No.: XM_208254), LOC284814 (Accession No.: XM_211651), LOC343718 (Accession No.: XM_293186), LOC339608 (Accession No.: XM_293187), LOC140164 (Accession No.: XM_071272), LOC348566 (Accession No.: XM_301875), POTE (Accession No.: NM_174981), LOC339612 (Accession No.: XM_293197), LOC350980 (Accession No.: XM_301877), LOC343720 (Accession No.: XM_293199),LOC348567 (Accession No.: XM_300378 LOC149777 (Accession No.: XM_084082), LOC339614 (Accession No.: XM_293168), LOC348568 (Accession No.:

XM_301868),LOC339616 (Accession No.: XM_295018 CYP4F3LP (Accession No.: AL163204), C21ORF81 (Accession No.: NM_153750), LOC339617 (Accession No.: XM_293171), LOC350981 (Accession No.: XM 04594), LOC339618 (Accession No.: XM_290963), RBM11 (Accession No.: NM_144770), ABCC13 (Accession Nos.: NM_138726,NM_172024, NM_172025, NM_172026), STCH (Accession No.: NM_006948), SAMSN1 (Accession No.: NM_022136), LOC343721 (Accession No.: XM_297865), LOC343722 (Accession No.: XM_293176), POLR2CP (Accession No.: AF127936), LOC350982 (Accession No.: XM_301873), LOC343724 (Accession No.: XM_297867), NRIP1 (Accession No.: NM_003489), LOC343725 (Accession No.: XM_297868), LOC348569 (Accession No.: XM_302829), LOC350983 (Accession No.: XM_304601), LOC343727 (Accession No.: XM_293178), LOC350984 (Accession No.: XM 04602), USP25 (Accession No.: NM_013396), LOC343728 (Accession No.: XM_293179), VDAC2P (Accession No.: AP001666), LOC343729 (Accession No.: XM_293180), LOC343730 (Accession No.: XMJ297870), LOC350985 (Accession No.: XM_304603), LOC343731 (Accession No.: XM_297871), LOC92876 (Accession No.: XM_047739), LOC343732 (Accession No.: XM_297872), LOC350986 (Accession No.: XM_304604), LOC350987 (Accession No.: XM 04605), LOC140190 (Accession No.: XM_071250), CXADR (Accession No.: NM_001338), BTG3 (Accession No.: NM 006806), LOC343733 (Accession No.: XM_297873), C21ORF91 (Accession No. : NM_017447), LOC150005 (Accession No.: XM_097795), LOC339619 (Accession No.: XM_297874), LOC343734 (Accession No.: XM_297875), CHODL (Accession No.: NM_024944), PRSS7 (Accession No.: NMJ302772), LOC348570 (Accession No.: XM_290329), LOC343735 (Accession No.: XM_297876), LOC343736 (Accession No.: XM_297877), SLC6A6P (Accession No.: AP001675), LOC350989 (Accession No.: XM_304606), C1QBPP (Accession No.: AP000568), LOC284819 (Accession No.: XM_210451), FDPSP (Accession No.: AP001678), LOC284820 (Accession No.: XM_210452), LOC254620 (Accession No.: XM_172114), RPS3AP (Accession No.: AP001679), LOC350990 (Accession No.: XM_304607), PPIAP (Accession No.: AP001680), NCAM2 (Accession No.: NM_004540), LOC343738 (Accession No.: XM_297878), LOC343739 (Accession No.: XM 297879), LOC343740 (Accession No.: XM_297880), C21ORF74 (Accession No.: NM_153203), LOC350991 (Accession No.: XM_304608), LOC343741 (Accession No.: XM_293182), MAPK6PS2 (Accession No.: AP000953), LOC254665 (Accession No.: XM 72116), LOC343742 (Accession No.: XM_297881), LOC343743 (Accession No.: XM_297882), ZNF299P (Accession No.: AP001686), LOC339621 (Accession No.: XM_293183), LOC343744 (Accession No.: XM_293184), TUBAP (Accession No.: AP000459), LOC343745 (Accession No.: XM_293185), LOC350992 (Accession No.: XM_304609), LOC339622 (Accession No.: XM_295016), LOC350993 (Accession No.: XM_304610), LOC284821 (Accession No.: XM_209374), C21ORF42 (Accession No.: NM_058184), MRPL39 Accession Nos.: NM_017446, NM_080794), JAM2 (Accession No.: NM_021219), ATP5J (Accession No.: NM_011512), GABPA (Accession No.: NM_002040), APP (Accession No.: NM_000484), LOC284822 (Accession No.: XM_210796), LOC343746 (Accession No.: XM_297883), LOC343747 (Accession No.: XM_297884), CYYR1 (Accession No.: NM_052954), LOC350994 (Accession No.: XM_304611), ADAMTS1 (Accession No.: NM_006988), ADAMTS5 (Accession No.: NM 007038), LOC150031 (Accession No.: XMJ 04501), GPXP2 (Accession No.: M93083), LOC343749 (Accession No.: XM_293192), LOC339623 (Accession No.: XM 93193), LOC343750 (Accession No.: XM_293194)₃ EIF4A1P (Accession No.: AP001604), RPL10P1 (Accession No.: AP001699), LOC284824 (Accession No.: XM_211652), LOC350995 (Accession No.: XM_304612), LOC284825 (Accession No.: XM_209376), LOC343751 (Accession No.: XM_297886), LOC343752 (Accession No.: XM_297887), C21ORF100 (Accession No.: NM_145033), LOC343753 (Accession No.: XM_297888), LOC343754 (Accession No.: XM_297889), LOC343755 (Accession No.: XM_297890), LOC150035 (Accession No.: XM_097793), N6AMT1 (Accession Nos.: NM _.013240, NM_182749), HSPDP7 (Accession No.: AF227510), ZNF294 (Accession No.: NMJ) 15565), RPL23P2 (Accession No.: AL163249), C21ORF6 (Accession No.: NM_016940), USP16 (Accession No.: NM_006447), CCT8 (Accession No.: NM_006585), C21ORF7 (Accession No.: NM 020152), C21ORF109 (Accession No.: NM_138968), BACH1 (Accession No.: NM_001186), C21ORF41 (Accession No.: NMJ38332), GRIK1 (Accession Nos.: NM_000830, NMJ 75611), LOC343758 (Accession No.: XM_297830), CLDN17 (Accession No.: NM_012131), CLDN8 (Accession No.: NM_011512), LOC350997 (Accession No.: XMJ01866), LOC343759 (Accession No.: XM 97831), LOC140256 (Accession No.: XM 71306), LOC339625 (Accession No.: XM 93163), LOC343760 (Accession No.: XM_293164), KRTAP13- 1 (Accession No.: XM_295015), LOC339626 (Accession No.: XM 93165), KRTAP19-1 (Accession No.: XM_295020), KRTAP8-1 (Accession No.: NM_175857), KRTAPl l-1 (Accession No.: NM_175858), LOC350998 (Accession No.: XMJ04592), LOC339627 (Accession No.: XM 97832), UBE3AP2 (Accession No.: AF010599), TIAM1 (Accession No.: NM_003253), LOC150051 (Accession No. XM_097792), FBXW1BP1 (Accession No.: AP000252), LOC339628 (Accession No. XM_293166), SOD1 (Accession No.: NM_000454), KIAA1172 (Accession No. XM_047889), HMG14P (Accession No.: AP000255), LOC348573 (Accession No. XMJ01867), HUNK (Accession No.: NM_014586), LOC343761 (Accession No. XM 97833), LOC150054 (Accession No.: XM_097797), C21ORF45 (Accession No. NM H8944), C21ORF108 (Accession No.: XMJ 14191), MGC14136 (Accession No.: NM_032910), C21ORF63 (Accession No.: NM_058187), LOC350999 (Accession No.: XMJ04593), FLJ10932 (Accession No.: NM 18277), TCP10L (Accession No. NM_144659), C21ORF59 (Accession No.: NM H7835), LOC343762 (Accession No. NM_293167), SYNJ1 (Accession No.: NM_003895), C21ORF66 (Accession Nos. NM 13329, NM_166631, NM_058191, NM_145328), C21ORF62 (Accession No. NM 19596), LOC343763 (Accession No.: XM_297834), LOC343764 (Accession No.: XM 87835), LOC284828 (Accession No.: XM_211650), OLIG2 (Accession No.: NM_005806), OLIG1 (Accession No.: XMJ 70977), LOC339629 (Accession No.: XM_295017), IFNAR2 (Accession No.: NM_000874), IL10RB (Accession No. NM_000628), IFNAR1 (Accession No.: NM 00629), LOC348574 (Accession No. XMJ 14184), LOC284829 (Accession No.: XM_211645), IFNGR2 (Accession No. NM 05534), C21ORF4 (Accession No.: NM_006134), RPS5L (Accession No. AP001717), C21ORF55 (Accession No.: NM 17833), GART (Accession Nos. NM 00819, NM_175085), SON (Accession Nos.: NM 03103, NM 32195, NM_058183, NM_138925, NM_138926, NM_138927), DONSON (Accession Nos.: NM H7613, NM_145794, NMJ 45795), CRYZL1 (Accession Nos.: NM_005111 NMJ45311, NM_145858), ITSN1 (Accession No.: NM_003024), LOC284831 (Accession No.: XMJ11644), ATP5O (Accession No.: NM 01697), LOC343766 (Accession No.: XM_297837), SLC5A3 (Accession No.: NM 06933), MRPS6 (Accession No.: NM 32476), C21ORF82 (Accession No.: NMJ53751), LOC343767 (Accession No.: XM 97838), LOC343768 (Accession No.: XM 97839), PRED37 (Accession No.: AA_613597), KCNE2 (Accession No.: NMJ72201), C21ORF51 (Accession No.: NM 58182), PRED38 (Accession No.: Q97G8-modell-riken), KCNE1 (Accession No.: NM_000219), DSCR1 (Accession No.: J^M_004414), PRED39 (Accession No.: AFJ46693), CLIC6 (Accession No.: NM_053277), LOC343769 (Accession No.: XM 97840), LOC343770 (Accession No.: XM_297841), RUNX1 (Accession No.: NM_001754), FLJ20856 (Accession No.: NM 25143), LOC140302 (Accession No.: XM 71234), LOC343771 (Accession No.: XM_297842), LOC351000 (Accession No.: XMJ04595), LOC343772 (Accession No.: XM 97843), LOC343773 (Accession No.: XM_297844), RPL34P3 (Accession No.: AF051934), LOC343774 (Accession No.: XM_297845), LOC343775 (Accession No.: XM_297846), LOC351001 (Accession No.: XMJ01870), LOC266693 (Accession No.: AF1015726), LOC343770 (Accession No.: XM_297847), RPS20P1 (Accession No.: AF015720), LOC284832 (Accession No.: XM_210450), LOC343777 (Accession No.: XM_293172), PPP1R2P2 (Accession No.: AF020802), LOC351002 (Accession No.: XMJ04596), RPL23AP3 (Accession No.: AB003151), LOC348576 (Accession No.: XMJ01872), RIMKLP (Accession No.: AP001724), C21ORF96 (Accession No.: AK_024509), c21ORF18 (Accession No.: NM_017438), C21ORF27 (Accession No.: AI685287), CBR1 (Accession No.: NM_001757), LOC284833 (Accession No.: XM_208252), CBR3 (Accession No.: NM_001757), LOC351004 (Accession No.: XMJ04597), RPL3P1 (Accession No.: AP001725), C21ORP5 (Accession No.: NM_011512), LOC257137 (Accession No.: XMJ 72106), NPX2 (Accession No.: NM_015358), CHAFIB (Accession No.: NM_005441), CLDN14 (Accession No.: NM_012130), LOC348577 (Accession No.: NMJ44492), LOC343780 (Accession No.: XM 97849), LOC343781 (Accession No.: XM_297850), LOC343782 (Accession No.: XM_293174), PSMD4 (Accession No.: AF50199), SIM2 (Accession No.: NM_005069, NM_009586), HLCS (Accession No.: NM_000411), DSCR6 (Accession No.: NM_018962), DSCR5 (Accession No.: NM_016430, NMJ53681, NMJ53682), TTC3 (Accession No.: NM_003316), DSCR9 (Accession No.: NMJ48675), DSCR3 (Accession No.: NM_006052), DYRK1A (Accession No.: NM_001396, NMJ01395, NM_130436, NMJ30437, NM_130438), LOC343783 (Accession No.: XM_297851), LOC343784 (Accession No.: XM_297852), KCNJ6 (Accession No.: NM_002240), LOC343785 (Accession No.: XM 97853), DSCR4 (Accession No.: NM_005867), DSCR8 (Accession No.: NM_032589), LOC343786 (Accession No.: XM_297854), LOC284834 (Accession No.: XM_209378), DSCR10 (Accession No.: NMJ48676), LOC343787 (Accession No.: XM_293175), C21ORF65 (Accession No.: NM_058188), KCNJ15 (Accession Nos.: NM_002243, NM_170736, NMJ70737), LOC343788 (Accession No.: XM_297855), ERG (Accession Nos.: NM_004449, NM_182918), LOC343789 (Accession No.: XM_297856), LOC343790 (Accession No.: XMJ297857), C21ORF24 (Accession No.: AI492145), ETS2 (Accession No.: NM_005239), LOC351005 (Accession No.: XMJ04598), LOC343793 (Accession No.: XM_297860), LOC343794 (Accession No.: XM 97861), LOC343795 (Accession No.: XM_297862), LOC348578 (Accession No.: XMJ04599), LOC140338 (Accession No.: XM_071256), LOC351006 (Accession No.: XMJ04600), PCBP2P1 (Accession No.: AF064859), DSCR2 (Accession No.: NM_003720), C21ORF107 (Accession No.: AJ002572), WDR9 (Accession Nos.: NM_018963, NM_033656), C21ORF87 (Accession No.: NMJ 53455), HMGN1 (Accession No.: NM_004965), WRB (Accession No.: NM_004627), MGC33295 (Accession No.: NMJ52505), C21ORF13 (Accession No.: AA405219), SH3BGR (Accession No.: NM_007341), LOC343797 (Accession No.: XM_297864), B3GALT5 (Accession Nos.: NM_006057, NMJ333170, NM 33171, NM_033172, NM_033173), LOC350084 (Accession No. XM 86761), IGSF5 (Accession No.: BG740428), PCP4 (Accession No. NM_006198), DSCAM (Accession No.: NM_001389), PRED42 (Accession No. P704J41D16_modellO-berlin), LOC284835 (Accession No.: XM_211649), BACE2 (Accession Nos.: NMJH2105, NM_138991, NM_138992), C21ORF75 (Accession No.: AI49439), FAM3B (Accession No.: NM_058186), MX2 (Accession No.: NM_002463), MX1 (Accession No.: NM_002462), TMPRSS2 (Accession No.: NM_005656), LOC348587 (Accession No.: XMJ04623), LOC284844 (Accession No.: XM_211662), FLJ32835 (Accession No.: NMJ52506), C21ORF20 (Accession No.: AW138631), C21ORF21 (Accession No.: AA969880), PRED76 (Accession No.: AK057397), C21ORF22 (Accession No.: AA435939), ANKRD3 (Accession No.: NM_020639), LOC343814 (Accession No.: XM_297901), PRDM15 (Accession No.: NMJ44771), LOC150138 (Accession No.: XM 86789), ZNF298 (Accession No.: NMJ 44771), C21ORF25 (Accession No.: XM_032945), LOC348588 (Accession No.: XMJ00789), LOC343815 (Accession No.: XM_297902), ZNF295 (Accession No.: NM_020727), LOC150142 (Accession No.: XM 86791), LOC284845 (Accession No.: XMJ11663), FLJ33471 (Accession No.: NM_152507), LOC348589 (Accession No.: XMJ01880), FLJ36335 (Accession No.: NMJ73568), UMODL1 (Accession No.: BM554176), ABCG1 (Accession Nos.: NM_004915, NM H6818), LOC150150 (Accession No.: XM 97820), LOC284846 (Accession No.: XMJ 10455), TFF3 (Accession No.: NM 03225), LOC343816 (Accession No.: XMJ97903), TFF2 (Accession No.: NM_005423), TFF1 (Accession No.: NM_003225), TMPRSS3 (Accession No.: NM 24022), UBASH3A (Accession No.: NM_018961), TSGA2 (Accession No.: NM_080860), SLC37A1 (Accession No.: NM 18964), LOC90620 (Accession No.: XM_032986), LOC343817 (Accession No.: XMJ93211), LOC255589 (Accession No.: XMJ73060), PDE9A (Accession No.: NM 02606), WDR4 (Accession Nos.: NM 18669, NM_033661, NM_033662), NDUFV3 (Accession No.: NM_021075), LOC90625 alias C21ORF105 (Accession Nos.: XM 33004, NM_000071, BC005107), PKNOX1 (Accession No.: NM 04571), CBS (Accession No.: NM 00071), LOC343818 (Accession No.: XMJ97904), U2AF1 (Accession No.: NM_006758), LOC343819 (Accession No.: XMJ97905), LOC343820 (Accession No.: XMJ97906), CRYAA (Accession No.: NM 00394), PRED47 (Accession No.: AW516786), PRED48 (Accession No.: AL530227), LOC256849 (Accession No.: XMJ73059), LOC351007 (Accession No.: XMJ04613), LOC351008 (Accession No.: XMJ04614), LOC343798 (Accession No.: XMJ97891), LOC351009 (Accession No.: XMJ01878), SNF1LK (Accession No.: NMJ73354), LOC150095 (Accession No.: XM_097805), PRED49 (Accession No.: AA707290), C21ORF72 (Accession No.: AA411910), C21ORF84 (Accession No.: NMJ53752), LOC343799 (Accession No.: XMJ97896), LOC351010 (Accession No.: XMJ04618), HSF2BP (Accession No.: NM_007031), RPL31P1 (Accession No.: AP001751), H2BFS (Accession No.: NM 17445), KIAA0179 (Accession No.: XM 35973), PDXK (Accession No.: NM 03681), MGC15873 (Accession No.: NM B2920), LOC91097 (Accession No.: XM_035977), C21ORF97 (Accession No.: NM 21941), CSTB (Accession No.: NM OOIOO), D212056E (Accession No.: NM 03683), LOC284837 (Accession No.: XMJ11658), LOC343800 (Accession No.: XMJ97892), MYL6P (Accession No.: AP001752), LOC343801 (Accession No.: XMJ97893), AGPAT3 (Accession No.: NM_020132), TMEMl (Accession No.: NM_03274), H2AFZP (Accession No.: AP001753), PWP2H (Accession No.: NM_005049), LOC284839 (Accession No.: XMJ11661), C21ORF33 (Accession No.: NM 04649), LOC351011 (Accession No.: XMJ04615), C21ORF32 (Accession No.: NM 175570), B7H2 (Accession No.: NM_015259), DNMT3L (Accession Nos.: NM 13369, NMJ75867), AIRE (Accession Nos.: NM_000383, NM_000658, NM_000659), PFKL (Accession No.: NM 02626), C21ORF2 (Accession No.: NM 04928), LOC343803 (Accession No.: XMJ97895), TRPM2 (Accession No.: NM_003307), LRRC3 (Accession No.: NM 30891), DKFZP434C128 (Accession No.: XM_036086), C21ORF30 (Accession No.: AL117578), C21ORF29 (Accession No.: NMJ 44991), C21ORF31 (Accession Nos.: AJ003549, AJ003550), PRED53 (Accession No.: BE797794), C21ORF90 (Accession No.: NMJ53204), IMMTP (Accession No.: AL773602), LOC348580 (Accession No.: XMJ04616), UBE2G2 (Accession Nos.: NM 03343, NMJ82688), SMT3H1 (Accession No.: NM 06936), LOC343804 (Accession No.: XMJ93202), PTTG1IP (Accession No.: NM 004339), LTGB2 (Accession No.: NM 000211), C21ORP69 (Accession Nos.: NM_058189, NM_182900), C21ORF67 (Accession No.: NM_058188), PRED55 (Accession No.: AJ003469), C21ORF70 (Accession No.: NM_058190), PRED74 (Accession No.: AI15287), LOC348581 (Accession No.: XMJ02830), LOC284840 (Accession No.: XMJ11657), LOC150112 (Accession No.: XM_086781), LOC343805 (Accession No.: XMJ93203), LOC351012 (Accession No.: XMJ04617), MGC10960 (Accession No.: NM 32653), ADARB1 (Accession Nos.: NM_001112, NM 15833), PRED58 (Accession No.: AA872876), C21ORF80 (Accession No.: NM 15227), PRED59 (Accession No.: BG221750), COL18A1 (Accession Nos.: NM 30582 NMJ30444, NMJ30445), C21ORF89 (Accession No.: NMJ53755), LOC284841 (Accession No.: XMJ11655), C21ORF86 (Accession No.: NMJ53454), C21ORF93 (Accession No.: NMJ 45179), LOC343806 (Accession No.: XMJ93204), LOC343807 (Accession No.: XMJ93205), COL18A1 (Accession Nos.: NM 30583, NMJ30444, NMJ30445), SLC19A1 (Accession No.: NM_003056), PRED61 (Accession No.: BI018918), LOC343809 (Accession No.: XM_297898), LOC339633 (Accession No.: XMJ93206), LOC150115 (Accession No.: XM_086780), LOC348582 (Accession No.: XMJ02831), LOC351013 (Accession No.: XMJ04619), LOC351014 (Accession No.: XMJ04620), LOC348583 (Accession No.: XMJ02833), LOC348584 (Accession No.: XMJ04621), PCBP3 (Accession No.: NM_020528), PRED62 (Accession No.: AJ003474), LOC351015 (Accession No.: XMJ04622), COL6A1 (Accession No.: NM_001848), LOC343811 (Accession No.: XM_297900), LOC343812 (Accession No.: XM_293207), LOC200292 (Accession No.: XMJ 17213), LOC339634 (Accession No.: XMJ93208), COL6A2 (Accession Nos.: NM 01849, NM_058174, NM_058175), FTCD (Accession No.: NM_006657), C21ORF56 (Accession No.: NM_032261), LSS (Accession No.: NM_002340), MCM3APAS (Accession No.: NM_018118), C21ORF85 (Accession No.: NMJ53753), MCM3AP (Accession No.: NM_003906), C21ORF57 (Accession No.: XM_059306), C21ORF58 (Accession No.: NM_032261), PCNT2 (Accession No.: NM_006031), LOC348585 (Accession No.: XMJ02832), C21ORF106 (Accession No.: BE791079), DIP2 (Accession No.: NM H5151), S100B (Accession No.: NM_006272), HRMT1L1 (Accession No.: NM 01535), LOC284843 (Accession No.: XM_209379) and RPL23AP4 (Accession No.: AP001478). RNA's particularly suitable as fetal marker RNA's include RNA's expressed from a gene within or near the Down's Syndrome Critical Region (DSCR) of human chromosome 21 selected from the group consisting of SLC5A3 (Accession No.: NM_006933), MRPS6 (Accession No.: NM_032476), C21ORF82 (Accession No.: NMJ 53751), LOC343767 (Accession No.: XM 97838), LOC343768 (Accession No.: XMJ97839), PRED37 (Accession No.: AA_613597), KCNE2 (Accession No.: NMJ72201), C21ORF51 (Accession No.: NM_058182), PRED38 (Accession No.: Q97G8-modell-riken), KCNE1 (Accession No.: NM_000219), DSCR1 (Accession No.: NM_004414), PRED39 (Accession No.: AFJ46693), CLIC6 (Accession No.: NM_053277), LOC343769 (Accession No.: XM 97840), LOC343770 (Accession No.: XMJ97841), RUNXl (Accession No.: NM_001754), FLJ20856 (Accession No.: NM_025143), LOC140302 (Accession No.: XM_071234), LOC343771 (Accession No.: XMJ97842), LOC351000 (Accession No.: XMJ04595), LOC343772 (Accession No.: XMJ97843), LOC343773 (Accession No.: XMJ97844), RPL34P3 (Accession No.: AF051934), LOC343774 (Accession No.: XMJ97845), LOC343775 (Accession No.: XMJ97846), LOC351001 (Accession No.: XMJ01870) LOC266693 (Accession No.: AF1015726), LOC343770 (Accession No.: XM_297847) RPS20P1 (Accession No.: AF015720), LOC284832 (Accession No.: XMJ10450) LOC343777 (Accession No.: XMJ93172), PPP1R2P2 (Accession No.: AF020802) LOC351002 (Accession No.: XMJ04596), RPL23AP3 (Accession No.: AB003151) LOC348576 (Accession No.: XMJ01872), RIMKLP (Accession No.: AP001724) C21ORF96 (Accession No.: AK_024509), C21ORP18 (Accession No.: NM_017438) C21ORP27 (Accession No.: AI685287), CBR1 (Accession No.: NM_001757) LOC284833 (Accession No.: XMJ08252), CBR3 (Accession No.: NM_001757) LOC351004 (Accession No.: XMJ04597), RPL3P1 (Accession No.: AP001725) C21ORF5 (Accession No.: NM_011512), LOC257137 (Accession No.: XMJ 72106) NPX2 (Accession No.: NM_015358), CHAFIB (Accession No.: NM_005441) CLDN14 (Accession No.: NM_012130), LOC348577 (Accession No.: NMJ44492) LOC343780 (Accession No.: XMJ97849), LOC343781 (Accession No. XMJ97850), LOC343782 (Accession No.: XMJ93174), PSMD4 (Accession No. AF50199), SIM2 (Accession No.: NM_005069, NM_009586), HLCS (Accession No. NM 000411), DSCR6 (Accession No.: NM_018962), DSCR5 (Accession No. NM H6430, NMJ53681, NMJ53682), TTC3 (Accession No.: NM_003316), DSCR9 (Accession No.: NMJ48675), DSCR3 (Accession No.: NM_006052), DYRK1A (Accession No.: NM_001396, NMJ01395, NMJ30436, NMJ30437, NMJ30438), LOC343783 (Accession No.: XM_297851), LOC343784 (Accession No.: XM_297852), KCNJ6 (Accession No.: NM_002240), LOC343785 (Accession No.: XMJ97853), DSCR4 (Accession No.: NM_005867), DSCR8 (Accession No.: NM 32589), LOC343786 (Accession No.: XM_297854), LOC284834 (Accession No.: XM 09378), DSCR10 (Accession No.: NMJ48676), LOC343787 (Accession No.: XMJ93175), C21ORP65 (Accession No.: NM_058188), KCNJ15 (Accession Nos.: NM_002243, NMJ70736, NMJ70737), LOC343788 (Accession No.: XMJ97855), ERG (Accession Nos.: NM_004449, NMJ82918), LOC343789 (Accession No.: XMJ97856), LOC343790 (Accession No.: XMJ97857), C21ORF24 (Accession No.: AI492145), ETS2 (Accession No.: NM_005239), LOC351005 (Accession No.: XMJ04598), LOC343793 (Accession No.: XM_297860), LOC343794 (Accession No.: XMJ97861), LOC343795 (Accession No.: XM 97862), LOC348578 (Accession No.: XMJ04599), LOC140338 (Accession No.: XM_071256), LOC351006 (Accession No.: XMJ04600), PCBP2P1 (Accession No.: AF064859), DSCR2 (Accession No.: NM_003720), C21ORF107 (Accession No.: AJ002572), WDR9 (Accession Nos.: NM_018963, NM 33656), C21ORF87 (Accession No.: NMJ 53455), HMGN1 (Accession No.: NM_004965), WRB (Accession No.: NM_004627), MGC33295 (Accession No.: NMJ52505), C21ORF13 (Accession No.: AA405219), SH3BGR (Accession No.: NM_007341), LOC343797 (Accession No.: XM_297864), B3GALT5 (Accession Nos.: NM_006057, NM_033170, NM_033171, NM_033172, NM_033173), LOC350084 (Accession No.: XM 86761), IGSF5 (Accession No.: BG740428), PCP4 (Accession No.: NM_006198), DSCAM (Accession No.: NM_001389), PRED42 (Accession No.: P704J41D16_modell0-berlin), LOC284835 (Accession No.: XMJ11649), BACE2 (Accession Nos.: NMJH2105, NMJ38991, NMJ38992), C21ORF75 (Accession No.: AI49439), FAM3B (Accession No.: NM 58186), MX2 (Accession No.: NM_002463), MX1 (Accession No.: NM_002462), TMPRSS2 (Accession No.: NM_005656), LOC348587 (Accession No.: XMJ04623), LOC284844 (Accession No.: XM_211662), FLJ32835 (Accession No.: NMJ52506), C21ORF20 (Accession No.: AW138631), C21ORF21 (Accession No.: AA969880), PRED76 (Accession No.: AK057397), C21ORF22 (Accession No.: AA435939), ANKRD3 (Accession No.: NM_020639), LOC343814 (Accession No.: XM_297901), PRDM15 (Accession No.: NMJ44771), LOC150138 (Accession No.: XM_086789), ZNF298 (Accession No.: NMJ44771), C21ORF25 (Accession No.: XM_032945), LOC348588 (Accession No.: XMJ00789), LOC343815 (Accession No.: XM_297902), ZNF295 (Accession No.: NM_020727), LOC150142 (Accession No.: XM_086791), LOC284845 (Accession No.: XM_211663), FLJ33471 (Accession No.: NMJ52507), LOC348589 (Accession No.: XMJ01880), FLJ36335 (Accession No.: NMJ 73568), UMODL1 (Accession No.: BM554176), ABCG1 (Accession Nos.: NM_004915, NM_016818), LOC150150 (Accession No.: XM_097820), LOC284846 (Accession No.: XMJ 10455), TFF3 (Accession No.: NM 03225), LOC343816 (Accession No.: XMJ97903), TFF2 (Accession No.: NM 05423), TFF1 (Accession No.: NM_003225), TMPRSS3 (Accession No.: NM 24022), UBASH3A (Accession No.: NM_018961), TSGA2 (Accession No.: NM 80860), SLC37A1 (Accession No.: NM H8964), LOC90620 (Accession No.: XM 32986), LOC343817 (Accession No.: XMJ93211), LOC255589 (Accession No.: XMJ73060), PDE9A (Accession No.: NM_002606), WDR4 (Accession Nos.: NM U8669, NM_033661, NM_033662), NDUFV3 (Accession No.: NM_021075), LOC90625 alias C21ORF105 (Accession Nos.: XM_033004, NM_000071, BC005107), PKNOX1 (Accession No.: NM 04571), CBS (Accession No.: NM_000071), LOC343818 (Accession No.: XMJ97904), U2AF1 (Accession No.: NM 06758), LOC343819 (Accession No.: XMJ97905), LOC343820 (Accession No.: XMJ97906), CRYAA (Accession No.: NM 00394), PRED47 (Accession No.: AW516786), PRED48 (Accession No.: AL530227), LOC256849 (Accession No.: XMJ73059), LOC351007 (Accession No.: XMJ04613), LOC351008 (Accession No.: XMJ04614), LOC343798 (Accession No.: XMJ97891), LOC351009 (Accession No.: XMJ01878), SNFILK (Accession No.: NMJ73354), LOC150095 (Accession No.: XM_097805), PRED49 (Accession No.: AA707290), C21ORF72 (Accession No.: AA411910), C21ORF84 (Accession No.: NMJ53752), LOC343799 (Accession No.: XMJ97896), LOC351010 (Accession No.: XMJ04618), HSF2BP (Accession No.: NM 07031), RPL31P1 (Accession No.: AP001751), H2BFS (Accession No.: NM 17445), KIAA0179 (Accession No.: XM 35973), PDXK (Accession No.: NM_003681), MGC15873 (Accession No.: NM 32920), LOC91097 (Accession No.: XM 35977), C21ORF97 (Accession No.: NM_021941), CSTB (Accession No.: NM_000100), D212056E (Accession No.: NM_003683), LOC284837 (Accession No.: XMJ11658), LOC343800 (Accession No.: XMJ97892), MYL6P (Accession No.: AP001752), LOC343801 (Accession No.: XMJ97893), AGPAT3 (Accession No.: NM_020132), TMEMl (Accession No.: NM 3274), H2AFZP (Accession No.: AP001753), PWP2H (Accession No.: NM 05049), LOC284839 (Accession No.: XMJ11661), C21ORF33 (Accession No.: NM_004649), LOC351011 (Accession No.: XMJ04615), C21ORF32 (Accession No.: NMJH75570), B7H2 (Accession No.: NM 15259), DNMT3L (Accession Nos.: NM_013369, NMJ75867), AIRE (Accession Nos.: NM_000383, NM_000658, NM 00659), PFKL (Accession No.: NM_002626), C21ORF2 (Accession No.: NM_004928), LOC343803 (Accession No.: XMJ97895), TRPM2 (Accession No.: NM 03307), LRRC3 (Accession No.: NM_030891), DKFZP434C128 (Accession No.: XM_036086), C21ORF30 (Accession No.: AL117578), C21ORF29 (Accession No.: NMJ 44991), C21ORF31 (Accession Nos.: AJ003549, AJ003550), PRED53 (Accession No.: BE797794), C21ORF90 (Accession No.: NMJ53204), IMMTP (Accession No.: AL773602), LOC348580 (Accession No.: XMJ04616), UBE2G2 (Accession Nos.: NM_003343, NMJ82688), SMT3H1 (Accession No.: NM_006936), LOC343804 (Accession No.: XMJ93202), PTTG1IP (Accession No.: NM_004339), ITGB2 (Accession No.: NM_000211), C21ORF69 (Accession Nos.: NM_058189, NMJ82900), C21ORF67 (Accession No.: NM_058188), PRED55 (Accession No.: AJ003469), C21ORF70 (Accession No.: NM_058190), PRED74 (Accession No.: AI15287), LOC348581 (Accession No.: XMJ02830), LOC284840 (Accession No.: XMJ11657), LOC150112 (Accession No.: XM_086781), LOC343805 (Accession No.: XMJ93203), LOC351012 (Accession No.: XMJ04617), MGC10960 (Accession No.: NM 32653), ADARB1 (Accession Nos.: NM_001112, NMJH5833), PRED58 (Accession No.: AA872876), C21ORF80 (Accession No.: NM 15227), PRED59 (Accession No.: BG221750), COL18A1 (Accession Nos.: NM_030582 NMJ30444, NMJ30445), C21ORF89 (Accession No.: NMJ53755), LOC284841 (Accession No.: XMJ11655), C21ORF86 (Accession No.: NMJ53454), C21ORF93 (Accession No.: NMJ45179), LOC343806 (Accession No.: XMJ93204), LOC343807 (Accession No.: XM 93205), COLI 8A1 (Accession Nos.: NM 30583, NMJ30444, NMJ30445), SLC19A1 (Accession No.: NM_003056), PRED61 (Accession No.: BI018918), LOC343809 (Accession No.: XMJ97898), LOC339633 (Accession No.: XMJ93206), LOC150115 (Accession No.: XM_086780), LOC348582 (Accession No.: XMJ02831), LOC351013 (Accession No.: XMJ04619), LOC351014 (Accession No.: XMJ04620), LOC348583 (Accession No.: XMJ02833), LOC348584 (Accession No.: XMJ04621), PCBP3 (Accession No.: NM_020528), PRED62 (Accession No.: AJ003474), LOC351015 (Accession No.: XMJ04622), COL6A1 (Accession No.: NM_001848), LOC343811 (Accession No.: XMJ97900), LOC343812 (Accession No.: XMJ93207), LOC200292 (Accession No.: XMJ 17213), LOC339634 (Accession No.: XMJ93208), COL6A2 (Accession Nos.: NM_001849, NM_058174, NM_058175), FTCD (Accession No.: NM_006657), C21ORF56 (Accession No. NM_032261), LSS (Accession No.: NM_002340), MCM3APAS (Accession No. NM_018118), C21ORF85 (Accession No.: NMJ53753), MCM3AP (Accession No. NM_003906), C21ORP57 (Accession No.: XM_059306), C21ORF58 (Accession No. NM 2261), PCNT2 (Accession No.: NM_006031), LOC348585 (Accession No. XMJ02832), C21ORF106 (Accession No.: BE791079), DIP2 (Accession No. NM H5151), S100B (Accession No.: NM_006272), HRMTILI (Accession No. NM_001535), LOC284843 (Accession No.: XMJ09379) and RPL23AP4 (Accession No.: AP001478). RNA's that are preferred as fetal marker RNA's include RNA's expressed from a gene selected from the group consisting of LOC90625 alias C21ORF105 (Accession Nos.: XM_033004, NM_000071, BC005107), PTTGIIP (Accession No.: NM_004339) and DSCR4 (Accession No.: NM_005867). These 3 RNA's may be detected as sequences comprised in SEQ ID NO.'s: 1, 2 and 3, respectively. The skilled person will understand that likewise the sequences complementary to SEQ ID NO.'s: 1, 2 and 3 and/or allelic variants of SEQ ID NO.'s: 1, 2 and 3 or their complements may be detected. Of these, particularly preferred for use in the methods of the present invention is an RNA expressed from LOC90625 (i.e. SEQ ID NO.: 1), in view of its placental expression that is detectable in maternal plasma already early in pregnancy and that is upregulated in trisomic placenta's. In a preferred embodiment of the invention, the quantity or concentration of the fetal marker RNA in a maternal blood sample is compared to the quantity or concentration of a second fetal RNA, i.e. a so-called fetal reference RNA. Preferably, the fetal reference RNA is an RNA expressed from a different chromosome than chromosome 21, and preferably also the fetal reference RNA are expressed in the placenta. More preferably, the placental reference RNA is an RNA that is of trophoblastic, in particular of extravillus origin, i.e. at least expressed in those types of placental cells. A preferred fetal reference RNA for quantification of the fetal marker RNA in the methods of the invention is an RNA expressed from a gene selected from the group consisting of the genes β-hCG (Accession No.: BC006290), MYST4 (Accession No.: NM H2330), PSG9 (Accession No.: NM_002784), PLAC1 (Accession No.: NM_021796) and HNRPH3 (Accession No.'s: NM_021644 and NM_012207). These reference RNA's may be detected as sequences comprised in SEQ ID NO.'s: 4, 5, 6, 7, 8 and 9, respectively. The skilled person will understand that likewise the sequences complementary to SEQ ID NO.'s: 4, 5, 6, 7, 8 and 9 and/or allelic variants of SEQ ID NO.'s: 4, 5, 6, 7, 8 and 9 or their complements may be detected. As a control for the quality of the extracted RNA, samples may also be subjected to an RT-PCR assay for HLA-G mRNA, which is expressed by both fetal and maternal tissues, i.e. respectively in trophoblast and lymphocytes (see Hviid et al., 1998, Hum. Immunol. 59: 87-98; and Kirszenbaum et al., 1994, Proc. Nat. Acad. Sci. USA 91: 4209-4213). For detection, reverse transcription, amplification and hybridization of fetal RNA in the methods of the invention unique primers and probes are designed based on the available sequences of expressed fetal RNA's in databases. A unique priming sequence preferably is a sequence that is suitable to serve as a primer-binding site for amplification primers in PCR. The length of the priming sequence may vary from 15 to 40, preferably from 18 to 30, more preferably from 20 to 25. A priming sequence preferably is optimized to meet a number of criteria for optimal use as PCR primer, such e.g. the absence of sequences that can form hairpins or other secondary structures. The priming sequence may bind only to a single site in the target nucleic acid sequence, i.e. the fetal RNA to be converted into cDNA and/or amplified. It may therefore be useful to test that the selected primer sequence does not demonstrate significant matches to sequences in the GenBank database (or other available databases). Furthermore, the T_m (also referred to as T_am,) of the primer may be optimized by analysis of the length and GC content of the primer. Such optimal priming sequences can be designed using a standard PCR-primer selection program such as "Primer Designer" version 2.0 (copyright 1990, 1991, Scientific and Educational software), "PrimerSelect" of the DNAStar.TM. software package (DNAStar, Inc.; Madison, Wis.), and "Oligo 4.0" (National Biosciences, Inc.). Particularly preferred primers for use in the methods of the invention include e.g. SEQ ID NO.'s: 10, 11, 12, 13, 14 and 15. In a preferred method of the invention, more than one fetal marker RNA is detected. In a further preferred method of the invention, the quantity of the one or more fetal marker RNA's is compared to the quantity of more than one fetal reference RNA. As described above, the fetal/placental marker and reference RNA's extracted from a maternal blood sample, or cDNA's derived therefrom, are preferably amplified in vitro. Applicable amplification assays include but are not limited to reverse transcriptase polymerase chain reaction (RT-PCR), ligase chain reaction, RNA and cDNA signal amplification methods including branched chain signal amplification, amplifiable RNA reporters, Q-beta replication, transcription-based amplification, boomerang DNA amplification, strand displacement activation, cycling probe technology, isothermal nucleic acid sequence based amplification (NASBA), other self sustained sequence replication assays, and other nucleic acid amplification assays as known in the art, and/or any variations or combinations thereof, performed in either qualitative or quantitative fashion. For example, the methods of the invention can utilize nucleic acid amplification methods as known in the art, such as but not limited to adapting those described by Edmands et al. (1994, PCR Methods Applic. 3: 317- 319); Abravaya et al. (1995, Nucleic Acids Res. 23: 675-682); Urdea et al. (1993, AIDS 7 (suppl 2): S11-S14); Kievits et al. (1991, J. Virological Methods 35: 273-286); and in WO97/35589. In preferred embodiments of the methods of the invention, fetal RNA is converted into cDNA using reverse transcriptase prior to in vitro amplification using methods known in the art. For example, a sample, such as 10 μl extracted plasma or serum RNA is reverse transcribed in a 30 μl volume containing 200 Units of Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Madison, Wis.), a reaction buffer supplied by the manufacturer, 1 mM dNTPs, 0.5 micrograms random hexamers, and 25 Units of RNAsin (Promega, Madison, Wis.). Reverse transcription is typically performed under an overlaid mineral oil layer to inhibit evaporation and incubated at room temperature for 10 minutes followed by incubation at 37°C for one hour. Alternatively, other methods well known in the art can be used to reverse transcribe the mammalian RNA to cDNA. There are numerous methods available in the art for detection of nucleic acids, any of which may be used in the methods of the invention for the qualitative or quantitative detection of the products amplified as described above. A preferred method uses gel electrophoresis, such as e.g. electrophoresis in agarose or polyacrjamide gels (see e.g. in Sambrook and Russel, 2001, In: "Molecular Cloning: A Laboratory Manual", 3^rd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, NY). As an alternative to ethidium bromide or other in gel detection methods, the amplified product can be transferred from the gel to a membrane by blotting techniques to be detected with a labeled probe. Amplified products may also be detected using immunological detection methods such as e.g. described by Landgraf et al. (1991, Anal. Biochem. 198: 86-91; 1991, Anal. Biochem. 193: 231-235), Coutlee et al. (1989, Anal. Biochem. 181: 96-105) and Bobo et al. (1990, J. din Micra 28: 1968- 1973) or elecfrochemiluminescence detection methods, such as described by Blackburn et al. (1991, Olin. Chem. 37: 1534-1539), or DiCesare et al. (1993, BioTechniques L5: 152-157), and all methods utilizing reverse dot blot detection technology (Saiki et al., 1989, Science 233: 1076-1078), and all methods utilizing high-performance liquid chromatography. More preferably, however, for quantitative detection of the amplified products in the methods of the present invention real time PCR is used. Real time PCR amplification allows the quantitative detection of the logarithmically increasing amount of PCR product in a specific PCR reaction. Three main real-time PCR machines are currently on the market: (1) The light cycler, developed by ROCHE (http://www.biochem.roche.com/lightcvcler/) (Wittwer et al. 1989, Nucleic Acids Res. 17: 4353-7; Wittwer et al., 1997, Biotechniques. 22: 176-81), (2) the Taqman (commercialized by Perkin Elmer-Applied Biosystems

(http://www.appliedbiosystems.com products/, generating information on the ABI- PRISM 7700, 7900HT, and 5700 machines), and the (3) iCycler commercialised by BIO-RAD (http ://www.bio-rad.com/iCycler (for an overview of characteristics of the respective machines, the reader is referred to a molecular biology tools website: www.mv.cιi/Molbioltoolsrtpcr.html. (See also Bustin, 2000, J. Mol. Endocrinol. 25: 169-93). All three technologies depend on a similar detection method that is based on the real-time detection during a PCR amplification, of a fluorescent signal, the strength of which is proportional to the specific PCR product that is amplified. Yet, the molecular basis that underlies the generation of a quantitative fluorescence signal that corresponds to the amount of the PCR product is different in these three technologies. The Light Cycler e.g., can be used with a double strand DNA (dsDNA) fluorophore that specifically interacts with ds-DNA but does not produce a fluorescent signal with single strand DNA. Thus with increasing amounts of ds-DNA, generated through PCR amplification of the template, an increasing level of fluorescence is generated, thereby allowing quantification. A disadvantage of this method is that the generation of the fluorescent signal does not involve any specificity for the nucleotide sequence that is amplified. As a consequence, any dsDNA molecule in the reaction mixture, including aspecific amplification products, will contribute to the signal, which will result in an overestimation of the specific amplification product. Other real-time detection techniques do depend on nucleotide sequence specific fluorescence. For the Light Cycler, a hybridization probe detection system has been set- up that utilizes fluorophore energy transfer between two fluorescent groups. Similarly, detection tools have been developed for the Taqman and iCycler machines: SYBR® green (Morrison et al., 1998, Biotechniques 24: 954-8, 960, 962), TaqMan® probes (inter alia DNA-binding dyes, molecular beacons, hydrolysis probes), Molecular Beacons® (Stratagene) (Tyagi and Kramer, 1996, Nat. Biotechnol. 14: 303-8), and others, that are based on variable physical characteristics of the compounds used and generate a quantitative fluorescence signal reflecting the logarithmically increasing DNA duplex molecule during the PCR reaction cycles that is either non-specific or sequence specific. The real time detection of mRNA's in various crude samples requires a sequence specific approach to avoid false-positive signals and quantification artefacts. Many research groups employ the real-time PCR quantification method for the differential expression profiling of RNA's expressed from specific genes in specific tissues or in cells grown under different conditions: Marcucci et al., 2001, Leukemia, 15:1072-80; Brieland et al., 2001, Infect Irnmun. 69: 5046-55; Efferth et al., 2001, Int. J. Oncol. 19: 367-71 ; Evans et al, 2001, J. Clin. Endocrinol. Metab. 86: 3097-107; Elmaagacli et al., 2001, Br. J. Haematol. ϋ3: 1072-5; Harness et al., 2001, J. Neural. Sci. 187: 7-16; Takano et al., 2001, Br. J. Cancer. 2001 85: 102-6; Reid et al., 2001, Proc. Natl. Acad. Sci. U S A. 98: 7552-7; Jeyaseelan et al., 2001, Nucleic Acids Res. 29:E58-8; Harsch et al., 2001, Am. J. Pathol. 158: 1985-90; Faneyte et al., 2001, Int. J. Cancer 93: 114-22; Haufroid et al., 2001, Clin. Chem. 47: 1126-9; Wang et al., 2001, Mol. Vis. 7: 89-94; Kipar et al., Vet. Immunol. Immunopathol. 78: 305-15; Seeger et al., 2001, Cancer Res. 61: 2517-22; Latil et al., 2001 Cancer Res. 61: 1919-26; Wellmann et al., 2001, Clin. Chem. 47: 654-60; Godfrey et al, 2000, J. Mol. Diagn. 2: 84-91; Amabile et al., 2001, Haematologica.86: 252-9; and Aerts et al., 2001, Ann. Oncol. 12: 39-46. These methods may be adapted for quantification of the fetal marker and reference RNA's in the methods of the invention, as has been done by Ng et al. (2003, supra). In a preferred embodiment of the method of the invention, the maternal blood sample is a sample obtained from a pregnant woman during early pregnancy, preferably the sample is obtained in the first trimester or the sample is obtained at least prior to week 17, 16, 15, 14, 13, 12, 11, 10, 9 or 8 of gestation. In a preferred embodiment of the method of the invention the number of copies of one or more fetal marker RNA's is determined in a maternal blood sample, e.g. using a real time quantitative RT-PCR as described above. Preferably the number of copies of one or fetal reference RNA's is also determined in the maternal blood sample. Preferably, the average value of the number of copies of a given fetal marker RNA in a given quantity of maternal blood sample from healthy control pregnancies is determined for a given age of gestation by normalizing the value to the average number of copies of the one or more fetal reference RNA's in the same blood samples. A Down's syndrome-affected pregnancy may then be diagnosed if the normalized number of copies of a given fetal marker RNA in the maternal blood sample is at least 1.1, 1.2, 1.4, or 1.8 times higher than the average value of that marker RNA for the healthy control pregnancies of (about) the same age of gestation. In a further aspect, the present invention relates to a "kit" containing elements for use in the methods of the invention as described above. Such a kit for prenatal detection, diagnosis, monitoring, or prediction of Down's syndrome-affected pregnancies may comprise a carrier to receive therein one or more containers, such as tubes or vials. The kit may further comprise unlabeled or labeled oligonucleotides (primers and/or probes) of the invention, e.g. to be used as primers, probes, which may be contained in one or more of the containers. The oligonucleotides may be present in lyophilized form, or in an appropriate buffer. One or more enzymes or reagents for use in reverse transcription and/or amplification reactions may further be contained in one or more of the containers. The enzymes or reagents may be present alone or in admixture, and in lyophilized form or in appropriate buffers. The kit may also contain any other component necessary for carrying out the present invention, such as buffers, enzymes, pipettes, plates, nucleic acids, nucleoside triphosphates, and gel materials. Such other components for the kits of the invention are known per se. The kit preferably at least provides for primers or probes for detection and or amplification of a fetal marker RNA, or cDNA derived therefrom, as describes above. The kit may further provide for the extraction of fetal RNA from maternal blood, plasma or serum. A preferred kit comprises primers and/or probes that hybridize to an RNA, or cDNA derived therefrom, expressed from a gene selected from the group consisting of LOC90625 (Accession No.: BC005107), PTTGIIP (Accession No.: NM_004339) and DSCR4 (Accession No.: NM_005867). Such primers or probes thus comprises sequences comprised in SEQ ID NO.'s: 1, 2 or 3, or their complements The kit may further provide for primers or probes for detection and/or amplification of a fetal reference RNA, or cDNA derived therefrom, as described above. A preferred kit comprises primers or probes used for the detection of an extracted fetal reference RNA, hybridize to an RNA, or cDNA derived therefrom, selected from the group consisting of RNA's expressed from a gene selected from the group consisting of the genes β-hCG (Accession No.: BC006290), MYST4 (Accession No.: NM_012330), PSG9 (Accession No.: NM_002784), PLAC1 (Accession No.: NMJ)21796) and HNRPH3 (Accession No.'s: NM 21644 and NMJ) 12207). The present invention is further illustrated by the following figures and examples, which, however, are not to be construed as limiting. The features disclosed in the foregoing description, in the following figures, examples and in the claims may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Description of the figures

Figure 1: Expression analysis in early human placenta of chromosome 21 -encoded genes. High RNA expression in early placental tissues and cells can be seen for LOC90625 (Fig 1A) similar in intensity to the chromosome 17-encoded control gene CSH1 (hPL) (Fig ID). Detectable signals are seen for the other chromosome 21- encoded genes, PTTGIIP (Fig IB) and DSCR4 (Fig 1C). Abbreviations: C: early placental tissue, S: extravillus trophoblast cell line, V: viUus fibroblast cells. MW: molecular weight marker (100 bp ladder).

Figure 2: Detection of CSH1 mRNA in maternal plasma samples. CSH1 cDNA amplicons generated by RT-PCR from RNA isolated from maternal plasma (weeks 9- 13) can be seen in all pregnant samples analyzed (lanes 4-10). All negative controls (n=8) of non-pregnant females were negative, only one is shown (lane 11) . Positive controls consisting of early placental tissues and cells are shown in lanes 1-3: 1. early placental tissue, 2. extravillus trophoblast cell line, 3. villus fibroblast cells. MW: molecular weight marker (100 bp ladder).

Figure 3 : Detection of chromosome 21 -encoded mRNA of placental origin in maternal plasma samples. LOC90625 cDNA amplicons generated by RT-PCR isolated from maternal plasma (week 9-13) are identified in all (n=8) pregnant samples analyzed (lanes 4-12). Note the absence of the non-specific lower bands with retention of the specific 362 bp band when RNA isolation was performed under vacuum controlled conditions (lanes 11-12). All negative controls of non-pregnant females (n=7) were negative, only one is shown (lane 13). Positive controls consisting of early placental tissues and cells are shown in lanes 1-3: 1. early placental tissue, 2. extravillus trophoblast cell line, 3. villus fibroblast cells. MW: molecular weight marker (100 bp ladder).

Examples

Example 1 Three chromosome 21 -encoded genes, e.g. LOC90625 (Accession No.: BC005107), PTTGIIP (Accession No.: NM_004339) and DSCR4 (Accession No.:

NM_005867) were tested for their expression and cell type distribution in early placental tissues. For comparison, expression analysis of CSH1 (hPL) was done identically. From early placental tissues and cells RNA was obtained and isolated as described (Oudejans et al., 2001, Genomics, 73: 331-337). These samples are representative of total chorionic villi, villus fibroblast cells and extravillus trophoblast cells (SGHPL5) (Cartwright et al, 2002 Placenta 23: 232-235). The latter cells were kindly provided by dr Judith Cartwright, London. The two-step, one-tube reverse transcription PCR assay was done as described (Oudejans et al., 2001, Genomics 73: 331-337) using the RNase-H negative Superscript II Platinum system (Life Technologies) in the presence of 1 M betaine. RT-PCR reactions were set up on ice within a PCR workstation (CBS Scientific). In brief, RNA was mixed with 50 pmol each of forward and reverse primers in a final volume of 10 μl in Micro Amp tubes and heated for 1 min at 95 °C followed by immediate cooling on ice. Forty microliters of master mix was subsequently added giving a final concentration of 1 x buffer, 1.25 mM magnesium sulphate, a 0.2 mM concentration each of dATP, dCTP, dGTP and dTTP, 1 M betaine (Fluka) and 1 μl of enzyme mix containing RNase H-negative Superscript II reverse transcriptase and Taq DNA polymerase (Life Technologies). Following reverse transcription for 30 min at 50°C, and denaturation for 1 min at 95°C, PCR was performed for 35 cycles (denaturation for 1 min at 95°C, annealing for 1 min and extension for 2 min at 72°C) followed by a final extension for 10 min at 72°C and cooling. All reactions were performed identically except that the annealing temperature was set at the temperature predicted to be optimal for each target (Oligo 4.0). Gene specific primers used were DSCR4-F 5'ATC TTG ACG AGA GAT GAT GAA CCC C-3', DSCR4-R 5'-TGT GGA GCC CAT GGA GGT AAA G -3' (Tann: 57°C), PTTGHP-F 5'-TGA AGA ACG TCT CCT GTC TT- 3', PTTG1IP-R 5'-ACT ACC GAC ATG GTG ATG AT-3' (Tann: 53°C) , LOC90625- F 5'-ACA CCG CCT CGT GTT GTC TGT TGG-3', LOC90625-R 5'TTG ACC CTG GTG CGA TGG ATC C -3' (Tann: 61 °C) HPL-F 5'-GCA CCA GCT GGC CAT TGA CA-3', HPL-R 5'-CCG TGC GGT TCC TCA GGA GTA T-3' (Tann: 58°C). For sequence analysis of amplified cDNA fragments, PCR products were following size separation by agarose electrophoresis, purified by affinity based isolation (Qiagen), subjected to cycle sequencing using BigDye terminators and analyzed using an ABI Prism 3100 Genetic Analyzer. Of the three chromosome 21 -encoded genes tested, LOC90625, PTTGIIP and DSCR4, all located within or near the Down syndrome critical region on chromosome 21q22, the strongest expression was seen for LOC90625 (Figure 1 A) with expression in all major cell components of the early human placenta, i.e. trophoblast, both villus and extravillus, as well as villus fibroblast. Expression of LOC90625 (Figure 1A) was similar in intensity to CSH1, although expression of the latter is restricted to trophoblast (Figure ID), while expression of LOC90625 is seen in all placental cells. Expression of PTTGIIP (Figure IB) and DSCR4 (Figure 1C) was easily detectable in early placenta but somewhat weaker as compared to LOC90625 and CSH1 when analyzed in weeks 8-12 of pregnancy.

Example 2 Subsequently it was tested whether chromosome 21 -encoded RNA from

LOC90625 could be identified in maternal plasma samples obtained from pregnant women in a similar gestational age window (weeks 9-13). Peripheral blood samples (n= 25) were collected with informed consent and approval of the Ethics Committee from pregnant women attending the Prenatal Diagnostic Unit of the Free University University Medical Center (Amsterdam). EDTA blood was collected between week 9-13 of pregnancy. All blood samples were obtained prior to invasive diagnostic procedures i.e. chorionic villus sampling. EDTA blood was stored at 4°C in upright position and processed within 24 hours after collection by 2 sequential centrifugation steps as described previously (Ng et al., 2003, Clin. Chem. 49: 727-731). In brief, following centrifugation for 10 min at 3000 rpm at 4°C in a Hettich Rotanta 96R centrifuge, plasma was subjected to a second centrifugation for 10 min at 14000 rpm at 4°C in a Hettich EBA12R centrifuge. Plasma was stored as aliquots at -70°C and thawed only once. Processing of blood was done within the laminar flow. RNA was extracted from 800 or 1600 μl of maternal plasma by silica-based affinity isolation using the QIAamp MinElute Virus Spin or QIAamp MinELute Virus Vacuum system (Qiagen, Hilden, Germany). Prior to isolation, plasma samples were thawed at RT, the heating block preheated to 56°C, carrier RNA added to AVE buffer (1 μg/μl), and protease thawed. All steps were done at room temperature (RT) unless stated otherwise. Fifty μl of protease (Qiagen) was added to a 1.5 ml tube, followed by addition of 400 μl of plasma, and 400 μl of buffer AL (with 28 μg/ml carrier RNA). Following vortexing for 15 sees, samples were incubated for 15 min at 56°C. Following centrifugation, 500 μl ethanol was added, and following vortexing for 15 sec, samples were left at room temperature for 5 min. All centrifugation steps were done for 1 min at 8000 rpm unless stated otherwise. Following centrifugation, the lysate mix was carefully loaded onto a QIAamp MinElute column and centrifuged. When the vacuum system was used, the columns were inserted into the QIAvac 24 vacuum manifold (QIAgen) according to the manufacturers instructions. The vacuum conditions used were -800 to -900 millibar with a 19L/min vacuumpump (Biometra MP26). All centrifugations steps except for the final elution step described below were substituted by processing with the vacuum system. For plasma starting volumes of 800 or 1600 μl, the number of tubes needed was increased accordingly, although the total volume was loaded onto a single column. Following transfer of the column to a new tube, 500 μl of buffer AW1 was added, followed by centrifugation. For on-column DNase digestion, 70 μl SDD buffer was added to 10 μl DNase, loaded onto the column and left for 15 min at RT. Following centrifugation, 500 μl of buffer AW2 was added, and samples recentrifuged. The column was placed into a new tube, followed by addition of 500 μl ethanol to the column and centrifugation. The column was placed into a new tube, and centrifuged for 3 min at 14000 rpm. The bound RNA was eluted by placing the column in a new tube, followed by application of 20-150 μl RNAse-free MilliQ water, incubation for 5 min at RT, and centrifugation for 1 min at 14000 rpm. Finally, samples were concentrated by using Microcon-PCR filters according to the manufacturers instructions (Millipore). The samples were used all-in-one i.e. the RNA obtained from 800 or 1600 μl plasma was used for a single RT-PCR. RT-PCR for LOC90625 and the reference RNA was performed as described in example 1. Amplified PCR products were subsequently visualized following size separation by agarose electrophoresis as described in example 1. For controls, plasma samples obtained from non-pregnant females of similar age and race were processed and used identically. The RNA isolation procedure was validated using CSH1 in a subset of the blood samples. In all pregnant females tested (n=7), CHS1 RNA could be detected easily (Figure 2) between weeks 9-13 using 800 μl of maternal plasma, while plasma specimens from non-pregnant females (n=7) were negative. No false-positives or false- negatives, respectively, were seen. Subsequently, RNA detection was performed identically for the chromosome 21- encoded gene LOC90625. Successful identification of mRNA from this gene in maternal plasma was seen, although with lower intensity compared to CSH1. When 800 μl plasma was used, detection was successful in 60%. When 1.6 ml plasma was used, detection of LOC90625 mRNA in first trimester plasma from pregnant females (n=8) was 100% (Figure 3). Moreover, sequence analysis of the LOC90625 cDNA amplicons generated in this way confirmed the specificity of the products.

Example 3 Peripheral blood samples are collected from pregnant women with informed consent and approval of the Ethics Committee. EDTA blood is collected between weeks 9 and 13 of pregnancy. All blood samples are obtained before invasive diagnostic procedures, i.e. chorionic villus sampling. Out of this cohort, a sample set of pregnant women (n=10) carrying a child with Down syndrome as proven independently by karyotyping of CVS samples is matched with a set of pregnant women with similar characteristics in age, pregnancy week and parity, carrying a child with normal karyotype (n=10). Following selection, the corresponding plasma samples are recovered from the plasma bank stored in the -70°C freezer, and given unique identification codes. The plasma samples are subsequently analytically processed by a person unaware of the clinical status of the samples being processed and without access to the identification codes. RNA is extracted as described in example 2. Subsequently, quantitative RT-PCR is performed for the chromosome 21 -encoded and reference RNA using real-time fluorescence resonance energy transfer technology (Light Cycler) including dual-color detection of target specific hybridization probes. Calibration is done with single stranded synthetic DNA identical and equal in length to the target RNA. Results are expressed as copy number per ml for both targets. Ratio's of copy number of chromosome 21 -encoded versus reference RNA are calculated for each sample and tested for the null hypothesis, assuming significantly increased ratio's in pregnant women carrying a Down syndrome baby versus normal matched controls. When the aforementioned procedure is followed for LOC90625 as an example of a chromosome 21 -encoded marker RNA the plasma samples taken from pregnant women carrying a child with Down syndrome show significantly higher expression levels of LOC90625 RNA than control plasma samples taken from pregnant women carrying a child with a normal karyotype. When the analysis is further extended with amplification of a reference RNA, selected from the genes β-HCG, MYST 4, PSG9, PLAC1 or HNRPH3, the ratio's of copy number of LOC90625 RNA versus reference RNA show the same pattern, i.e. significantly higher ratio's when the plasma samples are taken from pregnant women carrying a child with Down syndrome as compared to control plasma samples taken from pregnant women carrying a child with normal karyotypes. Prenatal diagnosis of a Down's syndrome pregnancy is thus feasible using either the expression level of LOC90625 or using its relative expression level in a ratio with at least one of reference RNA's taken from β-HCG, MYST 4, PSG9, PLAC1 or HNRPH3.

SEQUENCE LISTING SEQUENCE LISTING

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<221> misc_feature

<223> PTTGIIP (Accession No . : NM_004339)

<400> 2 ctgaacctca gccgcgccgg gaggcgagcc cgttggcgag gggcggggca gatggcgagg 60 gagcggggcg agctgctttt tgaggctgcg cactcgcatg gccccacctc gcaggtccac 120 ttccggcgcc gcggctgttc cgggcggaga ccgcttgtgc tggagtcgga gttgtaacgc 180 tccactgact gatagagcga ccggccgacc atggcgcccg gagtggcccg cgggccgacg 240 ccgtactgga ggttgcgcct cggtggcgcc gcgctgctcc tgctgctcat cccggtggcc 300 gccgcgcagg agcctcccgg agctgcttgt tctcagaaca caaacaaaac ctgtgaagag 360 tgcctgaaga acgtctcctg tctttggtgc aacactaaca aggcttgtct ggactaccca 420 gttacaagcg tcttgccacc ggcttccctt tgtaaattga gctctgcacg ctggggagtt 480 tgttgggtga actttgaggc gctgatcatc accatgtcgg tagtcggggg aaccctcctc 540 ctgggcattg ccatctgctg ctgctgctgc tgcaggagga agaggagccg gaagccggac 600 aggagtgagg agaaggccat gcgtgagcgg gaggagaggc ggatacggca ggaggaacgg 660 agagcagaga tgaagacaag acatgatgaa atcagaaaaa aatatggcct gtttaaagaa 720 gaaaacccgt atgctagatt tgaaaacaac taaagcgctc cagcacatca gtcccgacgc 780 ttcctgtgag gtgcacgctc cgcagcccag cccagccggg agaccacgtg gccattgcgg 840 tctcctgacc ttggccagtg aacctgccag ccttccagga caggcggccg gagagctgcc 900 cctgaaggac agtcctctcg tcttgcagac tggtgacctt ctattccctg ttcatctctg 960 tttctagatt tagtcacttg aaataagaaa tctttggggt ttgggctttt ttatactctt 1020 ctcagtttgt gaaacgctaa ctgcacacga agccgcctga cggcacccag cgctgtggct 1080 gtcattctcc cagggcagaa ccctgcgttt ctctctgtcc actaacaagc ttcacacgca 1140 acacagggaa gtcggtttga cttttgtcat gaggagaact gaccagccct catcattccc 1200 cataaaacca cggacagcgt ctgtgtgcgc atcttgagtc ttcacacctg ttgactcaca 1260 cggcttttgc tgatgacacg gggctccagt acacagtctg ataaggactt aacgtcctaa 1320 cctcaattgt attaaatagc attggggaat agctaaacct ttttaaaaaa atttattgga 1380 ttttcctccc tgcttaaaag atttcaccag aaaaccttca tataaaaatt caggcccttt 1440 ttggacaatt tttaaaattt gtatctttac tagaacatga gaatcttttt cccttggaag 1500 cttgaattat aaatgtggtg tttggcctgc ctcagcagca ccagttgact gctcgtgtgc 1560 cagcggtgtg gggaggacgg ggcaggacgc tgcagctctc tccagccctg ttggcatcct 1620 cagtgcctgc aggcctctcg ctgcctgttg ggctgtctgg ggggtggcca tttagggatc 1680 gtggggacgg ggtccacccc aagaagaaag aaaggcccgt ccacaggccc ggctctgggc 1740 cacgtgcccc ggaagcaggt gtgtccagag tcagctgagg gctctcccca caccacccag 1800 caggcgctgg tgctccttct gcctcatggg accagtccag cttccagccg ctctggctcg 1860 agggtggtct gagcacttcc ttctgagtgg gcttctctgg gagctctcca gtggcactgc 1920 tggacctgcc cacgtttctg taaaatcagg atacgtggct ttagtaagca gaccaagcgc 1980 ttcgtggcag ggaaagcagc gtgcggggaa gtcactgaaa agtgctgcct aaggaagttt 2040 ggaaatagtc cccgttccag attgccttga attttaaaac attttgcttt gggaaagtag 2100 gtcagcagca cctaagatca aggatgcgtt ccattttcac acttcacagt catgaaaact 2160 gagaagactg tcttcagcgt gaactaaagt tcacaggcag atcactgatc cagaacactt 2220 caagaactcg tcaaacagct cgataagcct ttttgactgt gtacatctgt accgggaata 2280 acattcctag gctgaaattt ccacaaagaa tagaacctgt acccagttct tcaggctgat 2340 ttccctgacc tcttgggcat ttgtatttgt agtaaagtat tgcagagatt cctaagtatt 2400 ttatagcagc catcaaaatt ggactttgta ttgtttattc ataaaagaca cttggtaata 2460 gacttcagtg aactctgtat gaatgcagta gtgtgcgtgc aaaatccgct tcctgagcgt 2520 agggtgctga gctggcgcta gggctcggtt gtgaaataca gcgtagtcag cccttgcgct 2580 cagtgtagaa acccacgtct gtaaggtcgg tcttcgtcca tctgcttttt tctgaaatac 2640 actaagagca gccacaaaac tgtaacctca aggaaaccat aaagcttgga gtgccttaat 2700 ttttaaccag tttccaataa aacggtttac tacctga 2737

<210> 3

<211> 1095 <212> DNA

<213> Homo sapiens

<220>

<221> misc_feature <223> DSCR4 (Accession No.: NM 005867)

<400> 3 cagtcacggg ccagcctttc ccttaaggat gtctgagagc tggtctcctc actcttttct 60 tccgcctatt aaactttccg cttcttaact cacccatgtg tgtccatgtc gttaatcatc 120 ttgacgagag atgatgaacc ccggatattt accccagaca gtgatgccgc ttcaccagca 180 ttgcactcta cttccccgct tcctgatcct gcctcagctt ctcctctcca cagagaagaa 240 aaaattctgc ctaaagtctg caacatcgtt tcctgcctga gtttcagcct gccagcttct 300 cctacggatt ctggacttgc cagccccaca atcataacca gagaggggca gcaattttgg 360 gcaaaatgtc tgatttggaa ataccaactt tacctccatg ggctccacaa gaaatcagat 420 gggagaaggg acaagcagat aagcgcaagc ccatcaacct gaaggcataa accacatcca 480 gccacctcct tctgatcagc agcaaagctg acgttttgat ctccatctgt ctgattcttg 540 tgtctacttc tcagtttaca actccagtgg gaaagaaaga gctttattta cagacccata 600 aaaatcccat cagtgtcgtc ccctgctgag aggccatgtg agaccatatg gaaaaacaac 660 agccataatg gcagcatggc agtggaaggg tttgtcttgt gcccaggcct tgcggtcatg 720 caagtttctt gtggatcctg ttgggaccag ccactcacca aggctgagta ggtccacaaa 780 taatggggac tttctaccag actcacagag aactgctggg tttttgggaa gggtgtgcgt 840 gtctttgggg catggaagtt ggggttatag tggagaccca gaggatgaga aaacttctct 900 gcctcagcag aagagtggca gctgagagag aggcaagaaa ctcgcaccca ctgtggactg 960 gggcagagag attttgagga gaatgaaatc cagaaactct gtgtggtatt agtttgtatc 1020 cagagggtga ccctcttctc aaggaaatgg gtgtcatcaa ttttctacac tattaaagat 1080 ataaagttct tggca 1095

<210> 4

<211> 85E <212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> β-hCG (Accession No. : BC006290)

<400> 4 caggacccca ccataggcag aggcaggcct tcctacaccc tactccctgt gcctccaggc 60 tcgactagtc cctagcactc gacgactgag tctctgagat cacttcaccg tggtctccgc 120 ctcacccttg gcgctggacc agtgagagga gagggctggg gcgctccgct gagccactcc 180 tgcgcccccc tggccttgtc tacctcttgc cccccgaagg gttagtgtcg agctcacccc 240 agcatcctac aacctcctgg tggccttgcc gcccccacaa ccccgaggta taaagccagg 300 tacacgaggc aggggacgca ccaaggatgg agatgttcca ggggctgctg ctgttgctgc 360 tgctgagcat gggcgggaca tgggcatcca aggagccgct tcggccacgg tgccgcccca 420 tcaatgccaσ cctggctgtg gagaaggagg gctgccccgt gtgcatcacc gtcaacacca 480 ccatctgtgc cggctactgc cccaccatga cccgcgtgct gcagggggtc ctgccggccc 540 tgcctcaggt ggtgtgcaac taccgcgatg tgcgcttcga gtccatccgg ctccctggct 600 gcccgcgcgg cgtgaacccc gtggtctcct acgccgtggc tctcagctgt caatgtgcac 660 tctgccgccg cagcaccact gactgcgggg gtcccaagga ccaccccttg acctgtgatg 720 acccccgctt ccaggactcc tcttcctcaa aggcccctcc ccccagcctt ccaagtccat 780 cccgactccc ggggccctcg gacaccccga tcctccσaca ataaaggctt ctcaatccgc 840 aaaaaaaaaa aaaaaaaa 858 <210> 5

<211> 6537 <212> DNA

<213> Homo sapiens

<220>

<221> misc_ofeature <223> MYST4 (Accession No.: NM 012330)

<400> 5 gataatctcc atttttgtca tgggactgtt aaaaacgttt ggaagttcca attctggtct 60 tgatttccca gttaaagatg ttctttcacc cgaatgcagt ctttcctgtt ggtaaaataa 120 gacaaccatc aacattgcct gtttgtctgc ttttgaatct cttaaggatg gatgtttgta 180 agatgttgct taatacagtc tggaatactc tgtccatttg ttgaattgta aatgactttc 240 aaatgtgcaa gttctgttaa atacaaagag aacctctatg ggtaactttt gtgttgaaga 300 agtcatttgt caaccatggt aaaacttgca aacccacttt atacagagtg gattcttgaa 360 gctatacaga aaataaaaaa gcaaaagcaa aggccctctg aagagagaat ctgccatgcg 420 gtcagtactt cccatgggtt ggataagaag acagtctctg aacagctgga actcagtgtt 480 caggatggct cagttctcaa agtcaccaac aaaggccttg cctcctataa ggacccagac 540 aaccctgggc gcttttcatc agttaaacca ggcacttttc ctaagtcagc caaggggtct 600 agaggatcat gtaatgatct ccgcaatgtg gattggaata aacttttaag gagagcaatt 660 gaaggacttg aggagccgaa tggctcctcc ctgaagaaca tagagaagta tctcagaagt 720 caaagtgatc tcacaagcac caccaacaac ccagcctttc agcagcggct gcgactgggg 780 gccaaacgcg ctgtgaataa tgggaggtta ctgaaagacg gaccgcagta cagggtcaat 840 tatgggagct tagatggcaa aggggcacct cagtatccca gtgcattccc atcctcgctc 900 ccacctgtca gccttctacc ccatgagaaa gaccagcccc gtgctgatcc cattccaata 960 tgtagcttct gtttggggac taaagaatca aatcgtgaaa agaaaccaga agaactcctc 1020 tcttgtgcag attgtggcag tagtggacac ccatcctgtt tgaaattttg tcctgaatta 1080 acaacaaatg taaaggcctt aaggtggcag tgcatcgaat gcaagacatg cagtgcctgt 1140 agagtccaag gcagaaatgc tgataatatg cttttttgtg attcctgtga tagaggattt 1200 catatggaat gctgtgaccc accactttcc agaatgccaa aagggatgtg gatttgccaa 1260 gtctgcagac caaagaaaaa gggaagaaaa ctacttcatg agaaagctgc acaaataaaa 1320 cgacgatatg caaaacccat tggacgaccg aaaaataaat taaagcaacg attgttgtct 1380 gtaaccagtg atgaaggatc catgaatgca ttcacaggaa gggggtcacc tggtaggggt 1440 caaaagacta aagtctgtac cacaccttca tctggtcatg ctgcatctgg gaaggactca 1500 agcagcagat tggctgttac agaccccact cggcctggtg ccaccaccaa aatcaccacc 1560 acctccacct acatttctgc ctctacactt aaagttaaca agaaaaccaa agggctcatt 1620 gatggcctta ctaagttttt tacaccatca cctgatggtc gcagatcacg aggtgaaatt 1680 atagactttt caaagcacta tcgtccaagg aaaaaggtct ctcagaaaca gtcatgcact 1740 tctcatgtgt tggctacagg taccacacaa aagctaaaac ctccaccttc ttcacttcca 1800 cccccaaccc ccatctccgg tcagagcccc agttcacaaa agtccagcac ggccacttct 1860 tctccctctc cccagagttc ttccagccag tgcagtgtgc cctccctgag cagccttacc 1920 actaacagcc agctgaaggc actctttgat gggctttctc atactatacc actcagggac 1980 agtctcgcaa aaagggacac ccσgagttat gcaccaccca aacgtatgcg tcgtaaaact gaattatctt ccacggcaaa atctaaagcc cacttctttg gcaaaagaga tattagaagt 2100 cggtttattt ctcactcctc ctcctctagc tgggggatgg ctagaggaag tatttttaaa 2160 gcaattgctc acttcaagcg aacaactttc cttaaaaagc acaggatgct aggcagatta 2220 aaatataaag tgacccctca gatggggacc ccctcaccag ggaaggggag cttgacagac 2280 ggaaggatta aacctgatca ggatgatgat actgaaataa aaataaacat caaacaagaa 2340 agtgcagatg taaatgtgat tggaaacaag gatgtcgtta ctgaagagga tttggatgtt 2400 tttaagcagg cccaggaact ttcttgggag aaaatagagt gtgagagtgg ggtggaagac 2460 tgtggccggt acccttctgt gattgaattt ggtaaatatg aaatccaaac ctggtactcc 2520 tcgccttacc cacaggaata tgcaagatta ccaaagcttt acctgtgtga attctgtctt 2580 aaatatatga aaagtaaaaa tattttgcta agacactcca agaagtgtgg atggtttcat 2640 cctccagcaa atgaaattta ccgaaggaaa gacctttcag tatttgaggt tgatgggaat 2700 atgagcaaaa tttattgcca aaacctttgc ttgttagcca agctcttcct ggaccacaaa 2760 acgttgtatt atgatgtcga gccattcctt ttttatgtcc ttacaaaaaa tgatgaaaag 2820 ggctgtcatc tggttggata cctctctaag gaaaagcttt gccagcagaa gtataatgtc 2880 tcctgcataa tgatcatgcc ccagcaccaa aggcaaggat ttggacggtt tctcattgat 2940 ttcagctatt tgctttctag aagagaaggc caagcagggt ctcctgaaaa gcctctctcc 3000 gatctgggcc gtctctccta cctggcatat tggaagagcg tcatcttgga gtatctctac 3060 caccaccatg agaggcacat cagcatcaag gcaattagca gagcgacggg catgtgccca 3120 catgacattg ccaccactct gcagcacctc cacatgatcg acaagagaga tggcagattt 3180 gtcatcatta gacgggaaaa gttgatattg agccacatgg aaaagctgaa aacctgttcc 3240 agagccaatg aacttgatcc agacagtctg aggtggaccc caattttaat ttctaatgct 3300 gcagtgtctg aagaagagcg agaagctgag aaagaggctg agcggctaat ggaacaagct 3360 agctgctggg agaaggagga acaagaaatc ctgtcaacta gagctaacag taggcaatca 3420 cctgcaaaag tacaatcgaa aaataaatat ttgcattcσc cggagagccg gccagtcaca 3480 ggggagcgag ggcagctgct ggagctgtct aaagagagca gtgaagaaga agaggaggag 3540 gaggacgagg aggaggaaga agaggaggaa gaagaggaag aggatgaaga ggaggaagaa 3600 gaggaagaag aagaagaaga agaagaaaat attcaaagct ctcccccaag attgacgaaa 3660 ccacagtcag ttgccataaa gagaaagagg ccttttgtac taaagaagaa aaggggtcgt 3720 aaacgcagga ggatcaacag cagtgtaaca acagagacca tttcagagac gacagaagta 3780 ctgaatgagc cctttgacaa ctcagatgaa gagaggccaa tgccacagct ggagcctacc 3840 tgtgagattg aagtggagga agatggcagg aagccagtcc tgagaaaagc attccagcat 3900 cagcctggga agaaaagaca aacagaggaa gaggaaggaa aagacaatca ttgcttcaag 3960 aatgctgacc cttgtagaaa caatatgaat gatgattcaa gtaacttgaa agaaggcagt 4020 aaagacaatc ccgaacctct aaagtgcaaa caagtgtggc caaaaggaac aaagcgcggt 4080 ctatctaagt ggaggcaaaa caaagagagg aagaccggat ttaaactgaa tttgtacacc 4140 ccgccagaaa cacccatgga gcctgacgag caggtaacag tggaagaaca gaaggagact 4200 tcagaaggaa aaaccagccc cagtcccatc aggattgagg aggaggtcaa ggaaactggg 4260 gaagccctgt tgcctcaaga ggaaaacaga agggaagaaa catgtgcccc tgtaagtcca 4320 aacacatcac caggtgaaaa accagaagat gatctcatca aacctgagga agaggaagag 4380 gaggaggagg aggaagagga agaagaggaa gaagaggaag gggaagaaga agaaggagga 4440 ggaaatgtag aaaaagatcc agatggtgct aaaagccaag aaaaagagga accagaaatc 4500 tccacggaaa aagaagactc tgcacgtttg gatgatcacg aagaggagga ggaagaggat 4560 gaagagccat cccacaacga ggaccatgat gccgatgacg aggatgacag ccacatggag 4620 tctgccgaag tggagaagga agagctgccc agagaaagct tcaaagaagt actggaaaac 4680 caggagactt ttttagacct taatgtgcag cctggtcact cgaacccaga ggtcttaatg 4740 gactgtggcg tcgacctgac agcttcttgt aacagtgagc ccaaggagct tgctggggac 4800 cctgaagctg tacccgaatc tgacgaggag ccacccccag gagaacaggc acagaagcag 4860 gaccaaaaga acagcaagga agtcgataca gagttcaaag agggaaaccc agcaaccatg 4920 gaaatcgact ctgagactgt ccaggccgtt cagtctttga cccaggagag cagcgaacag 4980 gacgacacct ttcaggattg tgccgagact caagaggcct gtagaagcct acagaactac 5040 acccgtgcag accaaagtcc acagattgcc accacgctcg acgattgcca acagtcggac 5100 cacagtagcc cagtttcatc cgtccactcc catcctggcc agtccgtacg ttctgtcaac 5160 agcccaagtg tccctgctct ggaaaacagc tacgcccaaa tcagcccaga tcaaagtgcc 5220 atctcagtgc catctctgca gaacatggaa accagtccca tgatggatgt cccatcagtt 5280 tcagatcatt cacagcaagt cgtagacagt ggatttagtg acctgggcag tatcgagagc 5340 acaactgaga actacgaaaa cccaagcagc tacgattcta ctatgggagg cagcatctgt 5400 ggaaacggct cttcacagaa cagctgctcc tatagcaacc tcacctccag cagtctgaca 5460 cagagcagct gtgctgtcac ccagcagatg tccaacatca gcgggagctg cagcatgctg 5520 cagcaaacca gcatcagctc ccctccgacc tgcagcgtca agtctcctca aggctgtgtg 5580 gtggagaggc ctccgagcag cagccagcag ctggctcagt gcagcatggc tgctaacttc 5640 accccaccca tgcagctggc tgaaatcccc gagacgagca acgccaacat tggcttatac 5700 gagcgaatgg gtcagagtga ttttggggct gggcattacc cgcagccgtc agccaccttc 5760 agccttgcca aactgcagca gttaactaat acacttattg atcattcatt gccttacagc 5820 cattccgctg ctgtgacttc ctatgcaaac agtgcctctt tgtccacacc attaagtaac 5880 acagggcttg ttcaactttc tcagtctcca cactccgtcc ctgggggacc ccaagcacaa 5940 gctaccatga ccccaccccc caacctgact cctcctccaa tgaatctgcc gccgcctctt 6000 ttgcaacgga acatggctgc atcaaatatt ggcatctctc acagccaaag actgcaaacc 6060 cagattgcca gcaagggcca catctccatg agaaccaagt cagcgtctct gtcaccagcc 6120 gctgccaccc atcagtcaca aatctatggg cgctcccaga ctgtagccat gcagggtcct 6180 gcacggactt taacgatgca aagaggcatg aacatgagtg tgaacctgat gccagcgcca 6240 gcctacaatg tcaactctgt gaacatgaac atgaacactc tcaacgccat gaatgggtac 6300 agcatgtccc agccaatgat gaacagtggc taccacagca atcatggcta tatgaatcaa 6360 acgccccaat accctatgca gatgcagatg ggcatgatgg gcacccagcc atatgcccag 6420 cagccaatgc agaccccacc ccacggtaac atgatgtaca cggcccccgg acatcacggc 6480 tacatgaaca caggcatgtc caaacagtct ctcaatggct cctacatgag aaggtag 6537

<210> 6

<211> 1731

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> PSG9 (Accession No.: NM 002784) <400> 6 agaaggagga aggacagcac agctgacagc cgtgctcaga cagcttctgg atcccaggct

60 catctccaca gaggagaaca cacaggcagc agagaccatg gggcccctcc cagccccttc 120 ctgcacacag cgcatcacct ggaaggggct cctgctcaca gcatcacttt taaacttctg 180 gaacccgccc accactgccg aagtcacgat tgaagcccag ccacccaaag tttctgaggg 240 gaaggatgtt cttctacttg tccacaattt gccccagaat cttcctggct acttctggta 300 caaaggggaa atgacggacc tctaccatta cattatatcg tatatagttg atggtaaaat 360 aattatatat gggcctgcat acagtggaag agaaacagta tattccaacg catccctgct 420 gatccagaat gtcacccgga aggatgcagg aacctacacc ttacacatca taaagcgagg 480 tgatgagact agagaagaaa ttcgacattt caccttcacc ttatacttgg agactcccaa 540 gccctacatc tccagcagca acttaaaccc cagggaggcc atggaggctg tgcgcttaat 600 ctgtgatcct gagactctgg acgcaagcta cctatggtgg atgaatggtc agagcctccc 660 tgtgactcac aggttgcagc tgtccaaaac caacaggacc ctctatctat ttggtgtcac 720 aaagtatatt gcaggaccct atgaatgtga aatacggaac ccagtgagtg ccagtcgcag 780 tgacccagtc accctgaatc tcctcccgaa gctgcccatc ccctacatca ccatcaacaa 840 cttaaacccc agggagaata aggatgtctt agccttcacc tgtgaaccta agagtgagaa 900 ctacacctac atttggtggc taaacggtca gagcctcccc gtcagtcccg gggtaaagcg 960 acccattgaa aacaggatac tcattctacc cagtgtcacg agaaatgaaa caggacccta 1020 tcaatgtgaa atacaggacc gatatggtgg cctccgcagt aacccagtca tcctaaatgt 1080 cctctatggt ccagacctcc ccagaattta cccttcattc acctattacc gttcaggaga 1140 aaacctcgac ttgtcctgct tcacggaatc taacccaccg gcagagtatt tttggacaat 1200 taatgggaag tttcagcaat caggacaaaa gctctttatc ccccaaatta ctagaaatca 1260 tagcgggctc tatgcttgct ctgttcataa ctcagccact ggcaaggaaa tctccaaatc 1320 catgacagtc aaagtctctg gtccctgcca tggagacctg acagagtctc agtcatgact 1380 gcaacaactg agacactgag aaaaagaaca ggctgatacc ttcatgaaat tcaagacaaa 1440 gaagaaaaaa actcaatgtt attggactaa ataatcaaaa ggataatgtt ttcataattt 1500 tttattggaa aatgtgctga ttctttgaat gttttattct ccagatttat gaactttttt 1560 tcttcagcaa ttggtaaagt atacttttat aaacaaaaat tgaaatattt gcttttgctg 1620 tctatctgaa tgccccagaa ttgtgaaact attcatgagt attcataggt ttatggtaat 1680 aaagttattt gcacatgttc caaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 1731

<210> 7

<211> 1131

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> PLAC1 (Accession No.: NM 021796)

<400> 7 ggcacgagga tcagaccatc agaaggattt gtataaagag tgactctcct atgaaggtaa 60 aggccacccc tcttcagttc cggtgactga gatacatttt tccaatcctg ggggcaaata 120 cagacacagc aagttccttc ttccctttgg aaatttggca gctgccttca ccagtgagca 180 caaagccaca tttcaaagga aactgacaaa ttatccccag ctgccagaag aagaaatcct 240 cactggacgg cttcctgttt cctgtggttc attatctgat tggctgcagg gatgaaagtt 300 tttaagttca taggactgat gatcctcctc acctctgcgt tttcagccgg ttcaggacaa 360 agtccaatga ctgtgctgtg ctccatagac tggttcatgg tcacagtgca ccccttcatg 420 ctaaacaacg atgtgtgtgt acactttcat gaactacact tgggcctggg ttgcccccca 480 aaccatgttc agccacacgc ctaccagttc acctaccgtg ttactgaatg tggcatcagg 540 gccaaagctg tctctcagga catggttatc tacagcactg agatacacta ctcttctaag 600 ggcacgccat ctaagtttgt gatcccagtg tcatgtgctg ccccccaaaa gtccccatgg 660 ctcaccaagc cctgctccat gagagtagcc agcaagagca gggccacagc ccagaaggat 720 gagaaatgct acgaggtgtt cagcttgtca cagtccagtc aaaggcccaa ctgcgattgt 780 ccaccttgtg tcttcagtga agaagagcat acccaggtcc cttgtcacca agcaggggct 840 caggaggctc aacctctgca gccatctcac tttcttgata tttctgagga ttggtctctt 900 cacacagatg atatgattgg gtccatgtga tcctcaggtt tggggtctcc tgaagatgct 960 atttctagaa ttagtatata gtgtacaaat gtctgacaaa taagtgctct tgtgaccctc 1020 atgtgagcac ttttgagaaa gagaaaccta tagcaacttc atgaattaag cctttttcta 1080 tatttttata ttcatgtgta aacaaaaaat aaaataaaat tctgatcgca t 1131

<210> 8 <211> 2316

<212> DNA

<213> Homo sapiens

<220> <221> misc feature <223> HNRPH3, transcript variant 2H9 (NM_012207)

<400> 8 ccgttgtggc gagcgctaca cgaggcaaac gacttctccc ttctttgaac tggaccccgc 60 gagcaccaga gtcggcgtaa ctatcgcctg acaggcattt aaatcaaacg gtattgagat 120 ggattgggtt atgaaacata atggtccaaa tgacgctagt gatgggacag tacgacttcg 180 tggactacca tttggttgca gcaaagagga aatagttcag ttctttcaag ggttggaaat 240 cgtgccaaat gggataacat tgacgatgga ctaccagggg agaagcacag gggaggcctt 300 cgtgcagttt gcttcaaagg agatagcaga aaatgctctg gggaaacaca aggaaagaat 360 agggcacagg tatattgaga tcttcagaag tagcaggagt gaaatcaaag gattttatga 420 tccaccaaga agattgctgg gacagcgacc gggaccatat gatagaccaa taggaggaag 480 agggggttat tatggagctg ggcgtggaag tatgtatgac agaatgcgac gaggaggtga 540 tggatatgat ggtggttatg gaggttttga tgactatggt ggctataata attacggcta 600 tgggaatgat ggctttgatg acagaatgag agatggaaga ggtatgggag gacatggcta 660 tggtggagct ggtgatgcaa gttcaggttt tcatggtggt catttcgtac atatgagagg 720 gttgcctttt cgtgcaactg aaaatgacat tgctaatttc ttctcaccac taaatccaat 780 acgagttcat attgatattg gagctgatgg cagagccaca ggagaagcag atgtagagtt 840 tgtgacacat gaagatgcag tagctgccat gtctaaagat aaaaataaca tgcaacatcg 900 atatattgaa ctcttcttga attctactcc tggaggcggc tctggcatgg gaggttctgg 960 aatgggaggc tacggaagag atggaatgga taatcaggga ggctatggat cagttggaag 1020 aatgggaatg gggaacaatt acagtggagg atatggtact cctgatggtt tgggtggtta 1080 tggccgtggt ggtggaggca gtggaggtta ctatgggcaa ggcggcatga gtggaggtgg 1140 atggcgtggg atgtactgaa agcaaaaaca ccaacataca agtcttgaca acagcatctg 1200 gtctactaga ctttcttaca gatttaattt cttttgtatt ttaagaactt tataatgact 1260 gaaggaatgt gttttcaaaa tattatttgg taaagcaaca gattgtgatg ggaaaatgtt 1320 ttctgtaggt ttatttgttg catactttga cttaaaaata aatttttata ttcaaaccac 1380 tgatgttgat actttttata tactagttac tcctaaagat gtgctgcctt cataagattt 1440 gggttgatgt attttactat tagttctaca agaagtagtg tggtgtaatt ttagaggata 1500 atggttcacc tctgcgtaaa ctgcaagtct taagcagaca tctggaatag agcttgacaa 1560 ataattagtg taactttttt ctttagttcc tcctggacaa cactgtaaat ataaagccta 1620 aagatgaagt ggcttcagga gtataaattc agctaattat ttctatatta ttatttttca 1680 aatgtcattt atcaggcata gctctgaaac attgatgatc taagaggtat tgatttctga 1740 atattcataa ttgtgttacc tgggtatgag agtgttggaa gctgaattct agccctagat 1800 tttggagtaa aaccccttca gcacttgacc gaaataccaa aaatgtctcc aaaaaattga 1860 tagttgcagg ttatcgcaag atgtcttaga gtagggttaa ggttctcagt gacacaagaa 1920 ttcagtatta agtacatagg tatttactat ggagtataat tctcacaatt gtattttcag 1980 ttttctgccc aatagagttt aaataactgt ataaatgatg actttaaaaa aatgtaagca 2040 acaagtccat gtcatagtca ataaaaacaa tcctgcagtt gggttttgta tctgatccct 2100 gcttggagtt ttagtttaaa gaatctatat gtagcaagga aaaggtgctt tttaatttta 2160 atccctttga tcaatatggc ttttttccaa attggctaat ggatcaaaat gaaacctgtt 2220 gatgtgaatt cagttattga acttgttact tgtttttgcc agaaatgtta ttaataaatg 2280 tcaatgtggg agataaaaaa aaaaaaaaaa aaaaaa 2316 <210> 9

<211> 2271

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> HNRPH3, transcript variant 2H9A (NM 021644)

<400> 9 ccgttgtggc gagcgctaca cgaggcaaac gacttctccc ttctttgaac tggaccccgc 60 gagcaccaga gtcggcgtaa ctatcgcctg acaggcattt aaatcaaacg gtattgagat 120 ggattgggtt atgaaacata atggtccaaa tgacgctagt gatgggacag tacgacttcg 180 tggactacca tttggttgca gcaaagagga aatagttcag ttctttcaag ggttggaaat 240 cgtgccaaat gggataaσat tgacgatgga ctaccagggg agaagcacag gggaggcctt 300 cgtgcagttt gcttcaaagg agatagcaga aaatgctctg gggaaacaca aggaaagaat 360 agggcacagg tatattgaga tcttcagaag tagcaggagt gaaatcaaag gattttatga 420 tccaccaaga agattgctgg gacagcgacc gggaccatat gatagaccaa taggaggaag 480 agggggttat tatggagctg ggcgtggaag ttatggaggt tttgatgact atggtggcta 540 taataattac ggctatggga atgatggctt tgatgacaga atgagagatg gaagaggtat 600 gggaggacat ggctatggtg gagctggtga tgcaagttca ggttttcatg gtggtcattt 660 cgtacatatg agagggttgc cttttcgtgc aactgaaaat gacattgcta atttcttctc 720 accactaaat ccaatacgag ttcatattga tattggagct gatggcagag ccacaggaga 780 agcagatgta gagtttgtga cacatgaaga tgcagtagct gccatgtcta aagataaaaa 840 taacatgcaa catcgatata ttgaactctt cttgaattct actcctggag gcggctctgg 900 catgggaggt tctggaatgg gaggctacgg aagagatgga atggataatc agggaggcta 960 tggatcagtt ggaagaatgg gaatggggaa caattacagt ggaggatatg gtactcctga 1020 tggtttgggt ggttatggcc gtggtggtgg aggcagtgga ggttactatg ggcaaggcgg 1080 catgagtgga ggtggatggc gtgggatgta ctgaaagcaa aaacaccaac atacaagtct 1140 tgacaacagc atctggtcta ctagactttc ttacagattt aatttctttt gtattttaag 1200 aactttataa tgactgaagg aatgtgtttt caaaatatta tttggtaaag caacagattg 1260 tgatgggaaa atgttttctg taggtttatt tgttgcatac tttgacttaa aaataaattt 1320 ttatattcaa accactgatg ttgatacttt ttatatacta gttactccta aagatgtgct 1380 gccttcataa gatttgggtt gatgtatttt actattagtt ctacaagaag tagtgtggtg 1440 taattttaga ggataatggt tcacctctgc gtaaactgca agtcttaagc agacatctgg 1500 aatagagctt gacaaataat tagtgtaact tttttcttta gttcctcctg gacaacactg 1560 taaatataaa gcctaaagat gaagtggctt caggagtata aattcagcta attatttcta 1620 tattattatt tttcaaatgt catttatcag gcatagctct gaaacattga tgatctaaga 1680 ggtattgatt tctgaatatt cataattgtg ttacctgggt atgagagtgt tggaagctga 1740 attctagccc tagattttgg agtaaaaccc cttcagcact tgaccgaaat accaaaaatg 1800 tctccaaaaa attgatagtt gcaggttatc gcaagatgtc ttagagtagg gttaaggttc 1860 tcagtgacac aagaattcag tattaagtac ataggtattt actatggagt ataattctca 1920 caattgtatt ttcagttttc tgcccaatag agtttaaata actgtataaa tgatgacttt 1980 aaaaaaatgt aagcaacaag tccatgtcat agtcaataaa aacaatcctg cagttgggtt 2040 ttgtatctga tccctgcttg gagttttagt ttaaagaatc tatatgtagc aaggaaaagg 2100 tgctttttaa ttttaatccc tttgatcaat atggcttttt tccaaattgg ctaatggatc 2160 aaaatgaaac ctgttgatgt gaattcagtt attgaacttg ttacttgttt ttgccagaaa 2220 tgttattaat aaatgtcaat gtgggagata aaaaaaaaaa aaaaaaaaaa a 2271

<210> 10

<211> 24

<212> DNA

<213> Artificial

<220>

<221> misc_feature

<223> Primer LOC90625-F

<400> 10 acaccgcctc gtgttgtctg ttgg

24

<210> 11

<211> 22

<212> DNA

<213> Artificial

<220>

<221> misc_feature

<223> Primer LOC90625-R

<400> 11 ttgaccctgg tgcgatggat cc 22

<210> 12 <211> 20 <212> DNA <213> Artificial

<220>

<221> misc_feature

<223> Primer PTTGIIP-F

<400> 12 tgaagaacgt ctcctgtctt

20

<210> 13

<211> 20

<212> DNA

<213> Artificial

<220>

<221> misc_feature

<223> Primer PTTGIIP-R

<400> 13 actaccgaca tggtgatgat 20

<210> 14

<211> 25

<212> DNA

<213> Artificial

<220>

<221> misc_feature <223> Primer DSCR4-F <400> 14 atcttgacga gagatgatga acccc 25

<210> 15 <211> 22

<212> DNA

<213> Artificial

<220> <221> misc_feature

<223> Primer DSCR4-R

<400> 15 tgtggagccc atggaggtaa ag

22

<210> 16

<211> 20

<212> DNA

<213> Artificial

<220>

<221> misc_feature <223> Primer HPL-F

<400> 16 gcaccagctg gccattgaca 20

<210> 17

<211> 22

<212> DNA

<213> Artificial <220> <221> misc_feature <223> Primer HPL-R

<400> 17 ccgtgcggtt cctcaggagt at

22

Claims

Claims

1. A method for prenatally diagnosing Down's syndrome, the method comprising: (a) detecting ex vivo a fetal marker RNA in a maternal blood sample; and, (b) providing the diagnosis based on at least one of the presence, quantity and/or concentration of the fetal marker RNA.

2. A method according to claim 1, whereby the fetal marker RNA is RNA transcribed from a gene on human chromosome 21.

3. A method according to claims 1 or 2, whereby the fetal marker RNA is RNA transcribed from a gene present within or near the Down's syndrome critical region on human chromosome 21.

4. A method according to any one of claims 1 - 3, whereby the fetal marker RNA is RNA expressed from LOC90625, PTTGIIP or DSCR4.

5. A method according to any one of claims 1 - 4, whereby in the maternal blood sample, the quantity or concentration of the fetal marker RNA is compared to the quantity or concentration of a fetal reference RNA.

6. A method according to claim 5, whereby the fetal reference RNA is RNA expressed from a different human chromosome than chromosome 21.

7. A method according to claim 6, whereby the fetal reference RNA is transcribed from a gene selected from the group consisting of β-hCG (Accession No.: BC006290), MYST4 (Accession No.: NMJU2330), PSG9 (Accession No.: NM__002784), PLAC1 (Accession No.: NM 21796) and HNRPH3 (Accession No.'s: NM 21644 and NM 12207).

8. A method according to any one of claims 1 - 7, whereby more than one fetal marker RNA is detected.

9. A method according to any one of claims 5 - 8, whereby quantity of the fetal marker RNA is compared to the quantity of more than one fetal reference RNA.

10. A method according to any one of claims 1 - 9, whereby the maternal blood sample is a sample obtained from a pregnant woman prior to week 17 of gestation.

11. A method according to any one of claims 1 - 10, whereby the fetal marker RNA and or the fetal reference RNA are expressed in the placenta.

12. A method according to any one of claims 1 - 11, whereby all nucleated and anucleated cell populations are removed from the blood sample prior to detection of the RNA.

13. A method according to claim 12, whereby the RNA is detected in maternal blood plasma or serum.

14. A method according to claim 13, whereby the RNA is detected in equal or less than 2 ml maternal blood plasma or serum.

15. A method according to any one of the preceeding claims, wherein the method comprises amplifying the RNA.

16. A method according to claim 15 wherein the RNA is converted into complementary DNA by a reverse transcriptase and then amplified by a polymerase chain reaction.

17. A kit for prenatal detection, diagnosis, monitoring, or prediction of a Down's syndrome-affected pregnancy, wherein the diagnostic kit provides for primers or probes used for the detection of an extracted fetal marker RNA, or cDNA derived therefrom, in a method according to any one of claims 1 - 17.

18. A kit according to claim 17, wherein the primers or probes hybridize to an RNA, or cDNA derived therefrom, selected from RNA's expressed from LOC90625, PTTGIIP, and DSCR4.

19. A kit according to claims 17 or 18, wherein the kit further provides primers or probes used for the detection of an extracted fetal reference RNA, or cDNA derived therefrom, in a method according to any one of claims 5 - 7 or 9.

20. A kit according to claims 19, wherein the primers or probes used for the detection of an extracted fetal reference RNA, hybridize to an RNA, or cDNA derived therefrom, selected from the group consisting of RNA's expressed from β-hCG (Accession No.: BC006290), MYST4 (Accession No.: NM_012330), PSG9 (Accession No.: NM_002784), PLAC1 (Accession No.: NM 21796) and HNRPH3 (Accession No.'s: NM 21644 andNM_012207).