WO2012101268A1 - In vitro method to determine the risk of a pregnant woman of carrying a fetus with fetal aneuploidy - Google Patents

Abstract

The present invention relates to the risk assessment of chromosomal aneupolidy in a fetus by the determination of purine nucleoside phophorylase (PNP) in a sample obtained from pregnant women.

Description

IN VITRO METHOD TO DETERMINE THE RISK OF A PREGNANT WOMAN OF CARRYING A FETUS WITH FETAL ANEUPLOIDY

Field of the invention

The present invention is in the field of clinical diagnostics. Particularly the present invention relates to the risk assessment of aneuploidy in a fetus by the determination of purine nucleoside phosphorylase (PNP) in a sample obtained from pregnant women.

Background of the invention

Chromosomal abnormality reflects an atypical number of chromosomes (aneuploidy) or a structural abnormality in one or more chromosomes. An extra or missing chromosome is a common cause of genetic disorders (birth defects) and occurs during cell division when the chromosomes do not separate properly between the two cells.

Every cell in the human body, apart from enucleated red blood cells and the haploid gametes, has 23 pairs of chromosomes (for a total of 46). One copy of each pair is inherited from the mother and the other copy is inherited from the father. The first 22 pairs of chromosomes (referred to as autosomes) are numbered from 1 to 22. The 23rd pair of chromosomes are the sex chromosomes. Normal females have two X chromosomes, while normal males have one X chromosome and one Y chromosome.

Chromosome abnormalities occur in 1 of 160 live births, the most common being extra chromosomes 21, 18 and 13 (Driscoll and Gross 2009, Clinical practice. Prenatal screening for aneuploidy. The New England Journal of Medicine 360 (24): 2556—62).

Most embryos cannot survive with a missing or extra autosome (numbered chromosome) and are spontaneously aborted. The most frequent aneuploidy in humans is trisomy 1 , although fetuses affected with the full version of this chromosome abnormality do not survive to term (it is possible for surviving individuals to have the mosaic form, where trisomy 16 exists in some cells but not all). The most common aneuploidy that infants can survive with is trisomy 21, also referred to as Down syndrome, which is affecting 1 in 800 births and occurs as a result of abnormal cell function due to the presence of an extra chromosome 21. Trisomy 18 (Edwards syndrome) affects 1 in 6,000 births, and trisomy 13 (Patau syndrome) affects 1 in 10,000 births. Only 10% of infants with trisomy 18 or 13 reach 1 year of age (Griffiths. Anthony JF: Miller, Jeffrey H; Suzuki, David T; Lewontin, Richard C; Gelbart, William M (2000). "Chromosome Mutation II: Changes in Chromosome Number". An Introduction to Genetic Analysis (7th ed.). New York: W. H. Freeman).

Monosomy refers to the lack of one chromosome of the normal complement. Partial monosomy can occur in unbalanced translocations or deletions, in which only a portion of the chromosome is present in a single copy. Monosomy of the sex chromosomes (45 ,X) causes Turner syndrome. Trisomy refers to the presence of three copies, instead of the normal two, of a particular chromosome. The presence of an extra chromosome 21 is found in Down syndrome. Trisomy 18 (Edwards Syndrome), trisomy 13 (Patau Syndrome), trisomy 12 (chronic lymphatic leukemia), trisomy 9 and trisomy 8 (Warkany Syndrome 2) are other autosomal trisomies recognized in live-born humans- Trisomy of the sex chromosomes is possible, such as in (47, XXX), called Triple-X- Syndrome or such as in (47, XXY), called Klinefelter ^'s Syndrome. Tetrasomy and pentasomy are the presence of four or five copies of a chromosome, respectively. Although rarely seen with autosomes, sex chromosome tetrasomy and pentasomy have been reported in humans, including XXXX, XXXXX, XXXXY and XYYYY (Linden M. et al 1995. "Sex chromosome tetrasomy and pentasomy". Pediatrics 96 (4 Pt 1): 672-82).

The incidence of fetal aneuplodies like Down syndrome, Edwards syndrome or Patau syndrome increases significantly with increasing maternal age. Since the 1970s, if a woman was 35 years or over at time of delivery, she was considered to be screen positive and was offered diagnostic procedures including amniocentesis or chorionic villus sampling (CVS) and karyotyping. With this approach, if 15% of pregnant women in a given population are > 35 years of age, approximately 40% of cases affected with Down syndrome will be detected with a false positive rate of 1 % (Summers et al. 2003. J Med Screen 10: 107-111). It has therefore been suggested that maternal age alone as a screening strategy for fetal aneuploidies is insufficient and should be abandoned. In addition, amniocentesis or chorionic villus sampling are invasive and involve risk for both, the woman and the fetus. For this and other reasons, amniocentesis or chorionic villus sampling and karyotyping are not routinely performed during all pregnancies.

At minimum, screening should allow for a detection rate of Down syndrome of 75% with no more than 5% of false positive results in women who are presenting in the first and second trimester (Summers et at. 2003, J Med Screen W: 107-111). There are a number of screening options that can be carried out in the first and/or second trimester (for review see: Summers et al. 2003. J Med Screen 10: 107-111\ The combination of maternal age, the serological markers pregnancy associated plasma potein A (PAPP-A) and free-beta human chorionic gonadotropin (beta-hCG) and the ultrasound marker nuchal translucency (NT) thickness has been demonstrated to function in week H-14 with a detection rate for Down syndrome of about 82 to 90% for a false positive rate of 5% (Malone et al. 2005. NEJM 353 (19): 2001-2010; Bindro et al. 2002. Ultrasound Obstet Gynecol 20: 219- 225). However, this screening still fail to detect a significant number of Down syndrome cases and other aneuploidy affected pregnancies and diagnoses still 5% falsely positive.

Alpha- fetoprotein (AFP), a protein produced by the fetal liver, and measured in maternal serum was identified in combination with maternal age as a second trimester marker. Further serum markers have subsequently been added, including human chorionic gonadotropin (hCG) which is elevated (Boeart et al. 1987. Prenat Diagn 7(9): 623-630), unconjugated estriol (uE3), which is decreased (Canick et al. 1988. Br J Obstet Gynaecol 95(4):330-333) and inhibin-A which is also elevated (Cuckle et al. 1996. Prenat Dia 16(12): 1095-1100). Wald et al. 1988 demonstrated that a combination of maternal age and the three maternal serum markers AFP, uE3 and hCG measured between 15 and 20 weeks' of gestation, would detect 65% of fetuses with Down syndrome with a false positive rate of 5% (Wald et al. 1988. BMJ 297: 883-887). By adding the measurement of the second trimester marker inhibin-A, resulting in a quad screen, the detection rate increases to approximately 75% to 80% of fetal DS with a false positive rate of 3 to 5% (Lambert-Messerlian et al. 1996. J Med Screen 3: 58-62). Again, such rates mean that the screening still fail to detect a significant number of Down syndrome and other aneuploidy affected pregnancies and these tests are limited to the second trimester. These screens thus suffer from the additional problem that once a risk of a genetic defect is predicted, and amniocentesis or another invasive prenatal definitive diagnostic procedure is performed to diagnose the genetic defect, such as Down syndrome, it is at an advanced date of gestation, when termination of a pregnancy can be more physically and emotionally trying for the mother, and when certain less traumatic abortion procedures, such as, vacuum curettage, may not be available.

Purine nucleoside phosphorylase (PNP) is a cytoplasmatic key enzyme (EC 2.4.2.1; UniProt accession number: P00491) involved in purine metabolism consisting of 289 amino acids in humans (SEQ-1D No. 1). PNP catalyzes the reversible phosphorolysis of N-ribosidic bonds of both nucleosides and deoxynucleosides, except adenosine, generating ribose (or desoxyribose) 1- phosphate and the corresponding purine base (Parks and Asarwal, in: P.P. Bover fed.), The Enzymes, Academic Press, New York, 1972. pp. 483-514). The major physiological substrates for mammalian PNP are inosine, guanosine and 2'-desoxyguanosine. PNP is specific for purine nucleosides in the β-configuration and exhibits a preference for ribosyl-containing nucleosides relative to the analogs containing arabinose, xylose and lyxose stereoisomers. Moreover, PNP cleaves glycosidic bonds with inversion of configuration to produce a-ribose 1 -phosphate, as shown by its catalytic mechanism (Porter 1992. J Biol Chem 267: 7342-7351).

PNP is found in a variety of organisms, both procaryotic and eucaryotic and has been localized in the capillary endothelium of the heart, lung and brain as well as in granulocytes, lymphocytes and erythrocytes (for review see: Moriwaki et al. 1999. Histol Histopathol 14: 1321-1340). The human erythrocyte enzyme is active as a homotrimer, with each subunit presenting a molecular weight of 32,000 Da (Lewis and Lowv 1979. J Biol Chem 254 (19): 9927-9932). Genetic deficiencies of PNP cause gradual decrease in T-cell immunity, though keeping B-cell immunity normal (Stoop et al. 1977. NEJM 296 (12): 651-655: Siegenbeek et al. 1977. Clin Chem Acta 74 (3): 271-279). Therefore inhibitors against PNP may have the potential to be used as therapeutic agents for treatment of immunological disorders, including type IV autoimmune disorders such as rheumatoid arthritis, psoriasis, inflammatory bowel disorders and multiple sclerosis as well as T-cell proliferative disorders such as organ transplant rejection, T-cell lymphoma and T-cell leukemia.

The activity of PNP enzyme was measured in blood as well as red blood cells and lymphocytes of patients with PNP deficiency (Siesenbeek et al. 1977. Clin Chem Acta 74 (3): 271-279: Osborne et al. 1977. Journal of Clinical Investigation 60: 741-746). Moreover, PNP activity has been measured in human amniotic fluid cells and chorionic villi for prenatal diagnosis of PNP deficiency (Kleijer et al. 1989. Prenatal Diagnosis 9: 401-407; Perignon et al. 1987. Journal of Pediatrics 111: 595-598). Ozer et al. discussed serum/ plasma PNP activity of treated humans as a marker to monitor drug-induced liver injury (Ozer et al. 2008. Toxicology 245:194-205).

Puukka et al. demonstrated a significant increase of PNP activity in isolated lymphocytes of patients with Down's syndrome (Puukka et al. 1986. Biochemical Medicine and Metabolic Biology 36: 45-50). However, the number of peripheral T-lymphocytes has been shown to be reduced in patients with Down's syndrome (Schlesinger et al. 1976. Advances in Experimental Medicine and Biology 66: 665-671) suggesting a decrease rather than an increase in lymphocytes PNP activity of patients with Down's Syndrome.

In contrast, nothing is known about the absolute concentration of circulating PNP in human blood and its relevance for the diagnosis of fetal aneuploidy in pregnant women. Thus, the inventors of the present invention have investigated whether the measurement of the level of purine nucleoside phosphorylase (PNP) and fragments thereof in a sample of a bodily fluid from a pregnant women could be used for the determination whether a pregnant woman has an increased risk of carrying a fetus with fetal aneuploidy.

Summary of the invention

The present invention relates to a method to determine whether a pregnant woman has an increased risk of carrying a fetus with fetal aneuploidy comprising the steps of:

I. providing a sample of a bodily fluid of said pregnant woman,

II. determining the level of PNP or fragments thereof and/or anti-PNP-antibody in said sample,

III. comparing said level with a reference level,

IV. identifying whether the level is different from said reference level and evaluating whether the fetus has an increased risk of fetal aneuploidy, if the level is different from the reference level and

V. wherein an increased level of PNP or fragments thereof and/or anti-PNP-antibody in comparison to said reference level is an indication of an increased risk of fetal aneuploidy wherein said fragments have a lengths of at least 6 amino acid residues.

Description of drawings

Figure 1 shows a silver- stained 2D-Gel of total secreted protein extract obtained from Trisomy 21 -affected cytotrophoblasts supernatant.

Figure 2 shows a segment of an immunoblot membrane of total secreted protein extract obtained from Trisomy 21 -affected cytotrophoblasts supernatant probed with serum from women with DS pregnancy. Figure 3 shows a PNP standard curve.

Figure 4 shows the correlation between PNP concentration and gestational age in women with normal pregnancy. Figure 5 is a box and whisker plot of PNP concentrations in women with normal and Down Syndrome pregnancy.

Figure 6 is a box and whisker plot of PNP multiple of the median (MoM) values in women with normal and Down Syndrome pregnancy.

Figure 7 shows ROC plots of single and combined markers for the diagnosis of Down Syndrome pregnancies (without gestational age as covariate).

Figure 8 shows ROC plots of single and combined markers for the diagnosis of Down Syndrome pregnancies (with gestational age as covariate).

Figure 9 shows ROC plots of single and combined markers (analyzing MoM values) for the diagnosis of Down Syndrome pregnancies. Figure 10 is a box and whisker plot of PNP concentrations in women with normal and Trisomy 13 pregnancy.

Figure 11 is a box and whisker plot of PNP multiple of the median (MoM) values in women with normal and Trisomy 13 pregnancy. Figure 12 shows ROC plots of single and combined markers for the diagnosis of Trisomy 13 pregnancies (without gestational age as covariate).

Figure 13 shows ROC plots of single and combined markers for the diagnosis of Trisomy 13 pregnancies (with gestational age as covariate).

Figure 14 shows ROC plots of single and combined markers (analyzing MoM values) for the diagnosis of Trisomy 13 pregnancies. Figure 15 is a box and whisker plot of PNP concentrations in women with nonnal and Trisomy 18 pregnancy.

Figure 16 is a box and whisker plot of PNP multiple of the median (MoM) values in women with normal and Trisomy 18 pregnancy.

Figure 17 shows ROC plots of single and combined markers for the diagnosis of Trisomy 18 pregnancies (without gestational age as covariate).

Figure 18 shows ROC plots of single and combined markers for the diagnosis of Trisomy 18 pregnancies (with gestational age as covariate).

Figure 19 shows ROC plots of single and combined markers (analyzing MoM values) for the diagnosis of Trisomy 18 pregnancies.

Detailed description of the invention

The present invention relates to a method to determine whether a pregnant woman has an increased risk of carrying a fetus with fetal aneuploidy comprising the steps of:

I. providing a sample of a bodily fluid of a pregnant women,

II. determining the level of PNP or fragments thereof and/or anti-PNP-antibody in said sample,

III. comparing said level with a reference level,

IV. identifying whether the level is different from said reference level and evaluating whether the fetus has an increased risk of fetal aneuploidy, if the level is different from the reference level,

V. wherein an increased level of PNP or fragments thereof and/or anti-PNP-antibody in comparison to said reference level is an indication of an increased risk of fetal aneuploidy wherein said fragments have a lengths of at least 6 amino acid residues.

According to the method, an increased risk of carrying a fetus with fetal aneuploidy in a pregnant woman is detected, when said detennined level of PNP and/ or fragments thereof is above a certain predetermined threshold level. Preferably, the predetermined threshold level of PNP is between 10 and 90 ng/mL, more preferred between 10 and 70 ng/mL, even more preferred between 10 and 50 ng/mL, most preferred between 10 and 35 ng/mL. In a preferred embodiment of the invention an increased risk of carrying a fetus with fetal aneuploidy in a pregnant woman is detected, when said determined level of PNP and/ or fragments thereof is higher than 90 ng/mL, preferably higher than 70 ng/mL, more preferably higher than 50 ng/mL, most preferred higher than 35 ng/mL.

In another embodiment of the invention, an increased risk of carrying a fetus with fetal aneuploidy in a pregnant woman is detected, when said determined level of PNP MoM is above a certain predetermined threshold level. Preferably, the predetermined threshold level of PNP MoM is between 0.6 and 6, more preferred between 0.6 and 4.5, even more preferred between 0.6 and 3.5, even more preferred between 0.6 and 2.5, most preferred between 0.6 and 1.0. In a preferred embodiment of the invention an increased risk of can'ying a fetus with fetal aneuploidy in a pregnant woman is detected, when said determined level of PNP MoM is higher than 6, preferably higher than 4.5, more preferably higher than 3.5, even more preferably higher than 2.5, most prefened higher than 1.0. In another embodiment of the invention further markers may additionally be determined selected from the group alpha-fetoprotein (AFP), unconjugated estriol (uE3), human chorionic gonadotropin (hCG), total hCG, free-alpha-hCG (alpha-hCG), free-beta human chorionic gonadotropin (beta-hCG), beta-core hCG, hyperglycosylated hCG (ITG), placental growth hormone (PGH), inhibin, dimeric inhibin-A (inhibin A), pregnancy- associated plasma protein A (PAPP-A), proform of eosinophilic major basic protein (proMBP), complexes of PAPP-A with proMBP (proform of major basic protein), ProMBP complexes with angiotensinogen and/or complement factors and split products, ADAM 12, cell -free fetal D A or RNA, or fragments thereof as well as ultrasound markers, nuchal translucency, femur length, absence of nasal bone, fetal malformations, maternal age, maternal history or gestational age.

In a preferred embodiment of the invention said further markers are selected from the group comprising AFP, hCG, uE3, PAPP-A, alpha-hCG, beta-hCG, ITG, proMBP, inhibin, inhibin A, ADAM 12, cell-free fetal DNA or RNA, ultrasound markers, nuchal translucency, femur length, absence of nasal bone, fetal malformations, maternal age, maternal history or gestational age.

In yet another preferred embodiment of the invention the multiple of the median (MoM) is determined. In another embodiment of the invention the measurement of PNP and/or anti-PNP-antibodies is carried out within the first to third trimester, more preferred within the first and second trimester (8^th to 26^th week of pregnancy), even more preferred within the first to early second trimester (8^th to 20^th week of pregnancy), even more preferred within the first trimester (8^th to 14^th week of pregnancy), mostly preferred with the early first trimester (8^th to 10^th week of pregnancy).

In another embodiment of the invention the measurement of PNP and/or anti-PNP-antibodies and/ or at least one further marker is carried out within the first to third trimester, more preferred within the first and second trimester (8^th to 26^th week of pregnancy), even more preferred within the first to early second trimester (8^th to 20* week of pregnancy), even more preferred within the first trimester (8^th to 14^th week of pregnancy), mostly preferred with the early first trimester (8* to 10* week of pregnancy).

First and second trimester markers can also be combined by integrated, sequential or contingent screening (Palomaki et al. 2009. Genetics in Medicine 11:669-881 included herein by reference in its entirety). Integrated screening refers to a two-stage process that integrates the most informative markers (from both the first and second trimesters) together into a single risk assessment. Integrated screening can be done for example using first- and second-trimester serum markers only (serum integrated test) or serum markers in combination with first-trimester nuchal translucency measurement (the full integrated test). Because screening results are reported in the early second trimester, the follow-up diagnostic procedure for screen-positive women is usually amniocentesis. Sequential (or step-wise) screening incorporates aspects of first-trimester and second-trimester screening in a two-step strategy, in an effort to preserve the benefits of each type of screening (early diagnosis of affected pregnancies and highest screening performance, respectively). The risk is calculated using information from both trimesters. Contingent screening is similar to sequential screening. However, in contingent screening, the first-trimester results are divided into three outcomes: screen-positive, screen-negative and intermediate/ pending risk. Those patients with intermediate risks will then provide a second- trimester sample for testing to compute an integrated risk. This strategy allows for early diagnosis of affected pregnancies among the small high-risk group (screen-positives) while concurrently offering early reassurance to the large low-risk group (screen-negatives).

In a preferred embodiment of the invention markers of the first and second trimester are combined by integrated, sequential and/or contingent screening.

In another preferred embodiment of the invention the biological fluid sample is selected from the group consisting of peripheral blood, serum, plasma, amniotic fluid and urine.

Preferably, these methods are used to evaluate whether the woman is carrying a fetus with an aneuploidy of chromosome 21, 18, 13, 12, 9, 8 or X, most preferred chromosome 21. The invention also relates to the use of the described methods and kits for the risk assessment of fetal aneuploidy in pregnant women.

A kit for determining the level of PNP or fragments thereof is used, wherein the kit comprises at least:

(i) a first capture molecule that specifically binds a first epitope of SEQ-ID No. 1.

In a preferred embodiment of the invention a kit for determining the level of PNP or fragments thereof is used, wherein the kit comprises at least:

(i) a first capture molecule that specifically binds a first epitope of SEQ-ID No. 1,

(ii) a second capture molecule that specifically binds to a second epitope of SEQ-ID No. 1, that is different from said first epitope. A kit for determining the level of anti-PNP-antibody or f agments thereof is used, wherein the kit comprises at least:

(i) a first capture molecule that specifically binds a first epitope of said anti-PNP- antibody.

In a preferred embodiment of the invention a kit for determining the level of anti-PNP-antibody or fragments thereof is used, wherein the kit comprises at least:

(i) a first capture molecule that specifically binds a first epitope of said anti-PNP- antibody,

(ii) a second capture molecule that specifically binds to a second epitope of said anti- PNP-antibody, that is different from said first epitope.

The presence of PNP and/or anti-PNP-antibodies can be measured or detected with relative ease in samples of bodily fluids (for example plasma, serum) using specific capture molecule assay techniques which are known to persons skilled in the art. Among specific capture molecules there are i.e. antibodies, aptamers, oligomers, antigens. Among specific capture molecule assay techniques there are standard antibody-based detection systems such as immunoassays, for example sandwich assays or competitive assays, immunoblotting techniques and quantitative mass spectrometry assays.

The preferred detection methods comprise immunoassays in various formats such as for instance radioimmunoassay (RIA), chemiluminescence- and fluorescence- immunoassays, Enzyme- linked immunoassays (ELISA), Luminex-based bead arrays, protein microarray assays, and rapid test formats such as for instance immuno chromatographic strip tests.

The assays can be homogenous or heterogeneous assays, competitive and non-competitive assays. In a particularly preferred embodiment, the assay is in the form of a sandwich assay, which is a non-competitive immunoassay, wherein the molecule to be detected and/or quantified is bound to a first antibody and to a second antibody. The first antibody may be bound to a solid phase, e.g. a bead, a surface of a well or other container, a chip or a strip, and the second antibody is an antibody which is labeled, e.g. with a dye, with a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general composition and procedures involved with "sandwich assays" are well-established and known to the skilled person (The Immunoassay Handbook. Ed. David Wild, Elsevier LTD. Oxford: 3rd ed. (May 2005). ISBN-13: 978- 0080445267; Hultschig C et al, Curr Omn Chem Biol. 2006 Feb;10(l):4-10. PMID: 16376134, incorporated herein by reference).

In a particularly preferred embodiment the assay comprises two capture molecules, preferably antibodies which are both present as dispersions in a liquid reaction mixture, wherein a first labelling component is attached to the first capture molecule, wherein said first labelling component is part of a labelling system based on fluorescence- or chemiluminescence-quenching or amplification, and a second labelling component of said marking system is attached to the second capture molecule, so that upon binding of both capture molecules to the analyte a measurable signal is generated that allows for the detection of the formed sandwich complexes in the solution comprising the sample.

Even more preferred, said labelling system comprises rare earth cryptates or rare earth chelates in combination with a fluorescence dye or chemiluminescence dye, in particular a dye of the cyanine type. In the context of the present invention, fluorescence based assays comprise the use of dyes, which may for instance be selected from the group comprising FAM (5-or 6- carboxyfluorescein), VIC, NED, Fluorescein, Fluoresceinisothiocyanate (FITC), IRD-700/800, Cyanine dyes, auch as CY3, CY5, CY3.5, CY5.5, Cy7, Xanthen, 6-Carboxy-2\4',7',4,7- hexachlorofluorescein (HEX), TET, 6-Carboxy-4',5'-dichloro-2ⁱ,7'-dimethodyfluorescein (JOE), N,N,N',N'-Tetramethyl~6-carboxyrhodamme (TAMRA), 6-Carboxy-X-rhodamine (ROX), 5-Carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), Rhodamine, Rhodamine Green, Rhodamine Red, Rhodamine 110, BODIPY dyes, such as BODIPY TMR, Oregon Green, Coumarines such as Umbelliferone, Benzimides, such as Hoechst 33258; Phenanthridines, such as Texas Red, Yakima Yellow, Alexa Fluor, PET, Ethidiumbromide, Acridinium dyes, Carbazol dyes, Phenoxazine dyes, Porphyrine dyes, Polymethin dyes, and the like. In the context of the present invention, chemiluminescence based assays comprise the use of dyes, based on the physical principles described for chemiluminescent materials in Kirk- Othmer, Encyclopedia of chemical technology, 4^th ed., executive editor, J. I. Kroschwitz; editor. M. Howe-Grant, John Wiley & Sons, 1993, vol.15, p. 518-562, incorporated herein by reference, including citations on pages 551-562. Preferred chemiluminescent dyes are acridiniumesters.

In a particular embodiment of the invention, the immunological reaction detection method is based on the so-called TRACE (Time Resolved Amplified Cryptate Emission) technology (Mathis, Clin Chem. (1993) 39(9). 1953-9). In the context of the present invention, capture molecules are molecules which may be used to bind target molecules or molecules of interest, i.e. analytes (in the context of the present invention PNP or fragments thereof and/or anti-PNP-antibodies and fragments thereof), from a sample. Capture molecules must thus be shaped adequately, both spatially and in terms of surface features, such as surface charge, hydrophobicity, hydrophilicity, presence or absence of lewis donors and/or acceptors, to specifically bind the target molecules or molecules of interest. Hereby, the binding may for instance be mediated by ionic, van-der-Waals, pi-pi, sigma-pi, hydrophobic or hydrogen bond interactions or a combination of two or more of the aforementioned interactions between the capture molecules and the target molecules or molecules of interest. In the context of the present invention, capture molecules may for instance be selected from the group comprising a nucleic acid molecule, a carbohydrate molecule, a PNA molecule, a protein, an antibody, a peptide or a glycoprotein. Preferably, the capture molecules or ligands are antibodies, including fragments thereof with sufficient affinity to a target or molecule of interest, and including recombinant antibodies or recombinant antibody fragments, as well as chemically and/or biochemically modified derivatives of said antibodies or fragments derived from the variant chain with a length of at least 12 amino acids thereof.

Aneuploidy is an abnormal number of chromosomes, and is a type of chromosome abnormality. An extra or missing chromosome is a common cause of genetic disorders (birth defects). Aneuploidy occurs during cell division when the chromosomes do not separate properly between the two cells.

Nuchal translucency (NT) is defined as "the subcutaneous collection of fluid in the fetal nuchal region," and its sonographic measurement in the late first trimester is a marker of risk of fetal Down syndrome. Bronshtein et al. and Szabo and Gellen first reported an association between increased NT in the first trimester and abnormal karyotype (Bronshtein et al. 1989.Am J Obstet Gvnecol 161:78-82; Szabo et al. 1990. Lancet 336:1133). By 1992, studies had demonstrated that NT measurement could serve as a useful screening marker of fetal Down syndrome (Nicolaides et al. 1992. BMJ 304:867- 869).

The identification of an absent nasal bone between 1 1 and 13 completed weeks' gestation is also reported to be a useful Down syndrome marker (Cicero et al. 2001. Lancet 358:1665-1667).

The term jjCOrrel ting", as used herein in reference to the use of diagnostic, prognostic and risk estimation marker(s), refers to comparing the presence or amount of the marker(s) in a pregnant woman to its presence or amount in a pregnant woman known to suffer from, or known to be at risk of, a given condition (e.g. carrying a fetus with fetal aneuploidy). A marker level in a pregnant woman^'s sample can be compared to a level known to be associated with a specific diagnosis. The sample^'s marker level is said to have been correlated with a diagnosis; that is, the skilled artisan can use the marker level to determine whether the pregnant woman suffers from a specific type diagnosis, and respond accordingly. Alternatively, the sample's marker level can be compared to a marker level known to be associated with a good outcome (e.g. the absence of disease etc.). In preferred embodiments, a panel of marker levels is correlated to a global probability or a particular outcome.

The term "level" in the context of the present invention relates to the concentration (preferably expressed as weight/ volume; w/v) of marker peptides taken from a sample of a pregnant woman. The term "sample" as used herein refers to a sample of bodily fluid obtained for the purpose of diagnosis, prognosis, or evaluation of a subject of interest, such as a pregnant woman. Preferred test samples include blood, serum, plasma, amniotic fluid and urine. In addition, one of skill in the art would realize that some test samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.

As mentioned herein in the context of proteins or peptides, the term fragment refers to smaller proteins or peptides derivable from larger proteins or peptides, which hence comprise a partial sequence of the larger protein or peptide. Said fragments are derivable from the larger proteins or peptides by saponification of one or more of its peptide bonds.

Gestation is the period of time between conception and birth during which the fetus grows and develops inside the mother's womb. Gestational age is the age of an embryo or fetus. In humans, a common method of calculating gestational age starts counting either from the first day of the woman's last menstrual period (LMP) or from 14 days before conception (fertilization). Counting from the first day of the LMP involves the assumption that conception occurred 14 days later. If the day of conception is known, the 14th day before conception is used in place of the LMP. Preferably the pregnant woman is within the first to third trimester, more preferred within the first to second trimester (8^th to 26^th week of pregnancy), even more preferred within the first to second trimester (8^th to 24^th week of pregnancy), even more preferred within the first to early second trimester (8^th to 20^th week of pregnancy), even more preferred within the first trimester (8^th to 14^th week of pregnancy), mostly preferred within the early first trimester (8^th to 10^th week of pregnancy).

A multiple of the median (MoM) is a measure of how far an individual test result deviates from the median. MoM is commonly used to report the results of medical screening tests, particularly where the results of the individual tests are highly variable. Concentrations of prenatal screening analytes change constantly throughout pregnancy. For example, AFP concentrations in maternal serum increase by about 15% per week during the most favorable time for detecting open neural tube defects (15 - 20^th week of gestation). Converting these values to a gestational age-specific median value (MoM) normalizes for this gestational age effect. A laboratory first obtains measures on sera obtained routinely from 300 to 500 women. Measurements are initially expressed in mass units (e.g. ng ml) or international units (e.g. lU/ml). Weighted log-linear regression analysis is used to calculate an equation to determine median levels for the analyte in question for each gestational week. Each women ^'s measurement is then divided by the median value for the appropriate gestational age resulting in a multiple of the median (MoM). The overall median value in a population of women is, by definition, 1.00 MoM.

As mentioned herein, an "assay" or "diagnostic assay" can be of any type applied in the field of diagnostics. Such an assay may be based on the binding of an analyte to be detected to one or more capture probes with a certain affinity. Concerning the interaction between capture molecules and target molecules or molecules of interest, the affinity constant is preferably greater than 10^s M^"1.

The term antibody generally comprises monoclonal and polyclonal antibodies and binding fragments thereof, in particular Fc~fragments as well as so called "single-chain-antibodies" (Bird et al. 1988. Science 242:423-6), chimeric, humanized, in particular CD -grafted antibodies, and dia or tetrabodies (Holliger et al 1993. Proc. Natl Acad. Sci. U.S.A. 90:6444-8). Also comprised are immunoglobulin like proteins that are selected through techniques including, for example, phage display to specifically bind to the molecule of interest contained in a sample. The sensitivity and specificity of a diagnostic and/or prognostic test depends on more than just the analytical "quality" of the test, they also depend on the definition of what constitutes an abnormal result. In practice, Receiver Operating Characteristic curves (ROC curves), are typically calculated by plotting the value of a variable versus its relative frequency in "normal" (i.e. apparently healthy) and "disease" populations (i.e. patients suffering from diabetes, insulin resistance and/or metabolic syndrome). For any particular marker, a distribution of marker levels for subjects with and without a disease will likely overlap. Under such conditions, a test does not absolutely distinguish normal from disease with 100% accuracy, and the area of overlap indicates where the test cannot distinguish normal from disease. A threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be abnormal and below which the test is considered to be normal. The area under the ROC curve (AUC) is a measure of the probability that the perceived measurement will allow correct identification of a condition. ROC curves can be used even when test results don't necessarily give an accurate number. As long as one can rank results, one can create a ROC curve. For example, results of a test on "disease" samples might be ranked according to degree (e.g. 1-low, 2=normal, and 3=high). This ranking can be correlated to results in the "normal" population, and a ROC curve created. These methods are well known in the art (See, e.g., Hanley et all 982. Radiology 143: 29-36). Preferably, a threshold is selected to provide a ROC curve area of greater than about 0.5, more preferably greater than about 0.7, still more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9. The term "about" in this context refers to +/- 5% of a given measurement.

The horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cut-off selected, the value of (1 -specificity) may be determined, and a corresponding sensitivity may be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test. The term "screening" in the context of the present invention refers to a process of surveying a population, using a specific marker or markers and defined screening cut-off levels, to identify the individuals in the population at higher risk for a particular disorder. Screening is applicable to a population (e.g. pregnant women); risk assessment is applied at the individual patient level. Maternal screening is based on selecting a subgroup of women who are at the highest risk of giving birth to a child with a chromosomal abnormality (e.g. aneuploidy). An individual woman has an a priori risk, which is e.g. an age-related risk, that is independent of maternal serum marker concentrations (Hormannsdorfer et al. 2009. Ultrasound Obstet Gynecol 33: 147-151).

The commonly used algorithm to assign a patient-specific risk utilizes the multiple of the median (MoM) results to calculate a likelihood ratio according to Palomaki and Haddow (Palomaki and Haddow 1987. Am J Obstet Gynecol 156: 460-463). The woman's measured value for the individual blood-derived marker is divided by the expected median value found in women with unaffected pregnancies at the same gestational age, to derive the MoM. The MoM values can be adjusted for other factors such as maternal weight, race and insulin- dependent diabetes mellitus. The probability that the (MoM) values for the combination of blood-derived markers tested belongs to the multivariate distribution of values found in unaffected pregnancies is calculated. The same calculation is performed by reference to the probability that the individual combination of values forms part of the multivariate distribution found in affected pregnancies. The ratio of the two probabilities is termed the likelihood ratio (LR) which indicates the likelihood that an individual woman has an affected pregnancy or not. The degree of separation between the multivariate distributions for affected and unaffected pregnancies changes with gestational age, i.e. there is a continuous change in the manner of calculating probability depending upon gestational age. This continuous change can be built into the algorithm used in the calculation.

Alternatively, newly developed "degrees of extremeness" (DoE) instead of MoM can be used for risk calculation with blood-derived markers (Merz 2007. Ultraschall in Med 28: 270-272: Schmidt et al. 2007. Frauenarzt 48: 1089-1092). The DoE is a ratio of the distance between the median value and actual value and the distance between the median value and 5^Λ percentile (when the measured value is below the median) or the distance between the median and the 95^th percentile (when the measured value is above the median) {Merz 2007. Ultraschall in Med 28: 270-272; Merz et al. 2007. Ultrasound Obstet Gynecol 30: 542-543). Under this assumption, a DoE is 0 at the median value, 1.0 at the 95^th percentile, and -1.0 at the 5^th percentile. Additionally, the Bayesian theorem (Schetinin et al. 2007. IEEE Trans Inf Techno! Biomed 11: 312-319) can be used instead of the mathematical concept of sequential likelihood ratios of Palomaki and Haddow (Palomaki and Haddow 1987. Am J Obstet Gynecol 156: 460-463). Subject of the present invention is also a method for determining a pregnant woman's risk of carrying a fetus with fetal aneuploidy in an analyzing system, the method, in an analyzing module of the analyzing system, comprising steps of:

receiving a measurement level of PNP or fragments thereof and/or anti-PNP antibodies,

receiving a measurement level of at least one further marker, and

determining a quantitative estimate of the risk of fetal aneuploidy by comparing the input levels of said markers with observed relative frequency distributions of marker levels in pregnancies affected with fetal aneuploidy and in unaffected pregnancies.

In a preferred embodiment of this method the step of determining the quantitative estimate comprises a step of deriving a likelihood ratio of fetal aneuploidy using a multivariate analysis of the measurement level of PNP or fragments thereof and/ or anti-PNP antibodies and a measurement level of at least one further marker based on distribution parameters derived from a set of reference data.

Preferably, the method further comprises a step of re-expressing each input level of a marker as a multiple of the median level and/ or as degrees of extremeness of the respective marker in unaffected pregnancies of the same gestational age as the fetus of the pregnant women, the re- expressed marker level being used in said determination of the risk of fetal aneuploidy.

More preferably, the method further comprising a step of comparing the quantitative estimate of risk with a predetermined cut-off level to classify the pregnant woman as screen-positive or screen-negative based on the comparison.

Another subject of the invention is a computer program product for determining a pregnant woman^'s risk of carrying a fetus with fetal aneuploidy on a computer system, the computer product comprising:

- receiving means for receiving a measurement level of PNP or fragments thereof and/or anti-PNP antibodies and a measurement level of at least one further marker, and

determination means for determining a quantitative estimate of the risk of fetal aneuploidy by comparing the input levels of said markers with observed relative frequency distributions of marker levels in pregnancies affected with fetal aneuploidy and in unaffected pregnancies.

As mentioned above, a woman has an a priori risk. This risk, which is preferably the age-specific risk, is modified by multiplying by the LR obtained for blood-derived markers to derive a combined risk. This combined risk may then be used to counsel the woman regarding the relative risk of fetal aneuploidy as opposed to the risk of miscarriage associated with subsequent diagnostic invasive procedure. In a preferred embodiment of the invention the measured one or more marker levels are adjusted for one or more factors selected from the group of maternal race, maternal weight, smoking status, prior pregnancy adverse outcome, multiple birth and diabetic status.

The estimated risk is classified as screen-positive or screen-negative based on a comparison with a pre-determined cut-off. The value of the cut-off may be altered to vary the detection rate and false-positive rate.

In one embodiment of the invention the pregnant woman is subjected to further testing e.g. high- resolution sonogram screening, amniocentesis or chorionic villus sampling when the pregnant woman was classified as screen-positive.

In a preferred embodiment of the invention an algorithm when executed on a computer causes the computer to perform a process for risk calculation, using the determination of an a priori e.g. age-related risk, the calculation of MoM or DoE of blood-derived marker concentrations, the mathematical concept of sequential likelihood ratios of Palomaki and Haddow or the Bayesian theorem.

Examples 1. Identification of PNP in T21 -affected placenta cytotrophoblasts as an immunogenic protein PNP as a first trimester maternal serum marker for Down's syndrome in first trimester: Development of a new homogenous sandwich fiuoroimmunoassay for PNP and clinical assessment on patients from Denmark. 1.2. Serum Samples a) Normal samples. Serum samples from first trimester pregnant women (n = 151) were obtained as part of a routine prenatal screening program for DS at Skejby University Hospital, Aarhus, Denmark. The program is specifically for women who are 8 to 13 weeks pregnant, and includes ultrasound examination.

b) DS samples. First trimester DS samples (n = 39) consisted of samples from the Skejby screening program. All DS diagnoses were established by karyotyping. Gestational age was determined by the date of the last menstrual period and, in most cases, confirmed by ultrasound examination.

All samples were stored at -80°C and were thawed prior to analysis of PNP.

1.3. Ethics

AH samples were either collected for projects approved by the Danish Central Biomedical Research Ethics Committee. 1.4. Ceil culture and sample preparation

Human placental choriocarcinoma cell line (JEG3) was purchased from Sigma and cultured according to the supplier's instruction. Villous cytotrophoblast culture was undertaken according to the procedure of Frendo (Frendo et ah, 2000). Cytotrophoblasts were isolated from T21- affected placentas (15 weeks of pregnancy) and cultured for 72h. For each culture, cells were plated on three dishes. Cells were switched from serum-supplemented medium into serum-free medium after 48h and incubated at 37°C, 5% C0₂ for 24h. Cell supernatant containing secreted proteins were then collected, passed through a 0.2 nm filter (Miilipore) to remove cell debris and dialyzed against 20 mmol/L Tris-HCl (pH 6.8). Samples were then precipitated with 80% ice- cold ethanol for 1 h, centrifuged at 38000g for 25 min at 4°C and precipitates were washed with 80% ethanol.

1.5. 2DE and immunoblotting

Ethanol precipitates were resuspended in isoelectro focusing medium containing 8 mol/L urea, 2% w/v CHAPS, 2% v/v Triton X-100, 8 g/L Pharmalyte 3-10 (GE Healthcare), 100 mmol/L dithiothreitol (DTT), 0.2% v/v Tergitol NP7 (Sigma) and traces of bromophenol blue (Laoudj- Chenivesse et al. 2002. Proteomics 2: 481-485). One hundred micrograms of total secreted protein (analytical gels) or 300 μ of total secreted protein for preparative gels were loaded onto nonlinear immobilized pH gradient (IPG) strips (Immobiline Dry-Strips, pH 3-10, 18 cm long, GE Healthcare). Isoelectric focusing was carried out with IPGphor (GE Healthcare) according to the manufacturer's instructions. After the first dimension, the IPG strips were equilibrated for 10 min at room temperature in a buffer containing 6 mol/L urea, 50 mmol/L Tris-HCl (pH 6,8), 300 g/L glycerol, 2% w/v sodium dodecyl sulphate (SDS), 10 g/L DTT and bromophenol blue and then for 15 min in the same buffer containing 15 g/L iodoacetamide instead of DTT. SDS- polyacrylamide gel electrophoresis (SDS-PAGE) was performed at 20 mA/gel with 12.5% polyacrylamide gels (20cm X 20cm X 1mm). The running buffer consisted of 25 mmol/L Tris, 192 mmol/L glycine, and 1 g/L SDS. The gels were silver stained according to the procedure of Shevchenko (Shevchenko et al. 1996) and gel images were scanned by a flatbed scanner.

For immunoblotting, proteins from 2DE gels were transferred onto nitrocellulose membranes (Bio-Rad Laboratories). After blocking 30 min in Tris-buffered saline (500 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 6.8)) supplemented with 3% gelatine (Prolabo), membranes were incubated 1 h at room temperature with serum pools [1 :100 dilution]. For serum pools, equal volumes of each of 6 DS samples (12+2 to 12+5 weeks of pregnancy) or 6 gestational age- matched unaffected pregnancy samples were used. The immunoproducts were visualized with a monoclonal anti-human IgG-alkaline phosphatase conjugate (Sigma) and with a NBT-BCIP colorimetric substrate (Pierce). Each wash and antibody dilution was performed with Tris- buffered saline supplemented with 0.05%) Tween 20 and 1% gelatine. Gel matching and spot evaluation were performed using Melanie II software (Gene-Bio). Immunoreactive spots that were positive with DS pregnancy serum compared to blots were probed with normal unaffected pregnancy serum in order to identify spots of interest.

1.6. Protein identification

The protein of interest was excised and digested in the gel by a sequencing grade of trypsin (Promega) according to the method of Shevchenko (Shevchenko et al, 1996). Digested peptides were mixed with the same volume of a-cyano-4-hydroxy-£ra«s-cirinamic acid (10 g/L in acetonitrilertrifluoroacetic acid, 50:0.1 %) and loaded on the target of a BIFLEX III MALDI-TOF mass spectrometer (Bruker-Franzen Analytik) using the Dry-droplet procedure (Karas et al., 1988). Spectra were analyzed using the XTOF software (Bruker-Franzen Analytik) and autoproteolysis products of trypsin (m/z 842.51 and 1045.56) were used as internal calibrates. The mass accuracy of our analyses was usually better than ±50 ppm. Tandem mass spectrometry (MS/MS) of selected peptides was performed on a quadrupole time-of-flight (Q-TOF) mass spectrometer (QSTAR; Sciex) equipped with a nanospray source (Protana Inc.).

1.7. Antibodies and recombinant protein

Recombinant human PNP was purchased from Calbiochem. For the generation of anti-PNP antibodies, 4 6-week-old female BALB/c mice were immunized with 50 μg human recombinant PNP dissolved in 11 mmol/L sodium phosphate buffer (pH 7.2) containing 140 mmol/L NaCl. Subsequent booster injections of immunogen were administrated in 4-week intervals. The fusion was done with X63 mouse myeloma cells (P3-X63-Ag8.653) 3 months after initial immunization. Two clones (OSS and 06S) were screened by homogenous fluoroimmunoassay as described in Section IMMUNOASSAY. The antibodies were purified by protein A Fast Flow affinity chromatography (GE Healthcare) according to the manufacturer's instructions.

1.8. Immunoassay

A homogenous sandwich fluoroimmunoassay using time resolved amplified cryptate emission (TRACE) technology (Mathis. 1993. Clin Chem 39(9): 1953-9) was developed for the detection of PNP. In the assay, purified anti-human PNP monoclonal antibodies of clones OSS and 06S were coupled to AF647 fluorophore (Molecular Probes Inc.) and to europium cryptate TBP-mono-MP (Cis Bio International), respectively. The coupling reactions were performed according to the manufacturer's prescribed coupling protocols.

The stock AF647-conjugated antibody and cryptate-conjugated antibody solutions were diluted at 5 g/mL and 0.35 g/mL with assay buffer [100 mmol/L sodium phosphate, 0.1% bovine serum albumin, 600 mmol/L KF, 0.2 mg/mL nonspecific mouse IgG, pH 7.1], respectively, prior to use. The culture supernatant of JEG3 cell line containing natural PNP was diluted in horse serum (Sigma) to give PNP standards. Standards were calibrated against highly purified recombinant human PNP (Calbiochem). The immunoassay was performed by incubating 14 μL· of samples/calibrators, 68 Ε of AF647-conjugated antibody solution and 68 μΐ_^ of cryptate- conjugated antibody solution at 37°C on BRAHMS ryptor automate (Cezanne SAS), according to the manufacturer's instructions. The reaction time of the assay was 19 min. The specific fluorescence (RFU) was measured by simultaneous dual wavelength measurement at 665 and 620 nm using a BRAHMS Kryptor automat.

Hybridoma cell line screening was performed using cryptate-conjugated goat anti-mouse IgG (Sigma) and AF647-conjugated recombinant human PNP diluted at 0.3 μg/mL and 2 μ /mL with assay buffer, respectively. 1.9. Statistics

Mathematical algorithms to determine DS risk are calculated by detemiining the medians for the normal population and the Down syndrome population. Multiples of the median (MoM) are used in order to standardize the findings. Logistic regression models and ROC analysis were performed for single markers and marker combinations. Sensitivity (the proportion of actual positives which are correctly identified as such by a biomarker) and specificity (proportion of negatives which are correctly identified) were calculated for selected cut-offs.

Multiple regression modelling of log transformed marker concentrations was used to obtain log MoM values specific to gestational age. Screening performance results are based on a patient- specific risk calculated from the product of the maternal age-related risk and the likelihood-ratio for the appropriate combination of biochemical markers. Where nuchal translucency (NT) has been included in the screening algorithm, the patient-specific risks have been calculated by multiplying the maternal age-related risk by the likelihood-ratio for the biochemistry and the likelihood-ratio for NT, as defined in the mixture-model (Wright et al. 2008. Ultrasound Obstet Gynecol 31: 376-383).

2. Clinical assessment of PNP as maternal serum marker for Trisomy 13 and Trisomy 18 in first trimester

a) Normal samples. Serum samples from first trimester pregnant women (n = 248) were obtained as part of a routine prenatal screening program for DS at King's College Hospital, London, UK. The program is specifically for women who are 1 1 to 14 weeks pregnant, and includes ultrasound examination.

b) Trisomy 13 samples. First trimester Trisomy 13 samples (n = 30) consisted of samples from the King's College Hospital screening program, All Trisomy 13 diagnoses were established by karyotyping.

c) Trisomy 18 samples. First trimester Trisomy 18 samples (n = 30) consisted of samples from the King's College Hospital screening program. All Trisomy 18 diagnoses were established by karyotyping.

Gestational age was determined by the date of the last menstrual period and, in most cases, confirmed by ultrasound examination. All samples were stored at -80°C and were thawed prior to analysis of PNP.

Results

1. Identification of PNP as serum marker for Down's syndrome in first trimester

To identify proteins reacting with IgG molecules in sera obtained from pregnant women carrying a fetus with and without DS, total secreted protein extract obtained from T21 -affected cytotrophoblasts supernatant was subjected to 2DE combined with immunoblotting using serum sample pools obtained from 6 pregnant women carrying a DS and from 6 women with unaffected pregnancies. When cytotrophoblast supernatant was separated by two-dimensional gel electrophoresis (2DE), staining of gels with silver revealed a pattern of hundreds of protein spots (Fig. 1). The placenta cytotrophoblasts proteins were transferred to nitrocellulose and probed with sera from women carrying a fetus with or without DS in order to identify immunoreactive spots of interest.

The patterns of placenta cytotrophoblasts antigens recognized by sera from women carrying a fetus with or without DS were studied by two-dimensional gel electrophoresis and western blotting. Comparison of patterns of antigens stained on 2DE western blots of sera from women carrying a fetus with DS and women with unaffected pregnancies revealed a difference. One spot (Fig. 1 and Fig. 2) was positive with DS pregnancy sera. This spot, with an apparent mass of 32 kDa and pi of 6.5, was identified as purine nucleoside phosphorylase (PNP, Swiss Prot Accession No. P00491) using a peptide mass fingerprinting method. Placenta cytotrophoblasts PNP reacted exclusively with sera from women carrying a fetus with DS (Fig. 2) but not with sera from women with unaffected pregnancies. This finding demonstrated the presence of anti- PNP antibodies in sera from women carrying a fetus with DS.

2. Clinical assessment of PNP on patients with Down syndrome pregnancy

To determine whether PNP is a useful marker for Down syndrome, the homogenous sandwich fluoroimmunoassay for PNP described above was used to evaluate PNP in serum samples from 151 women who underwent normal pregnancies and 39 samples from pregnant women carrying a DS. The reaction time of the assay was 19 min. The linearity of the assay for cell supernatant PNP was obtained between 4 and 606 ng/mL (Fig. 3).

There was no correlation between PNP concentration and gestational age (p>0.05) in women with normal pregnancy (Fig. 4). PNP concentrations in women with normal pregnancy ranged between 1.7 and 86.4 ng mL with a median concentration of 14.2 ng/mL (Fig. 5). In women with DS pregnancy PNP serum concentrations were significantly elevated when compared to women with normal pregnancy (p<0.0001) with measuring values between 4.3 and 196.1 ng/mL and a median of 40.4 ng/mL. The respective values of PNP MoM (multiple of the median) are shown in Fig. 6. PNP MoM in women with normal pregnancy ranged between 0.12 and 6.2 with a median of 1.0, whereas in women with DS pregnancy PNP MoM values were significantly elevated when compared to women with normal pregnancy (p<0.0001) ranging between 0.31 and 13.7 and a median of 2.6.

The weekly median MoMs for PNP, PAPP-A and free beta-hCG of women with DS pregnancy are presented in Table 1. There was no correlation between MoM PNP and MoM free beta hCG and between MoM PNP and MoM PAPP-A in the first trimester, respectively (p > 0.05). Results of logistic regression analysis for single and multimarker models of the marker concentration levels (with and without gestational age as a covariate) are presented in Table 2. ROC plot analysis for single markers without inclusion of gestational age as a covariate revealed an area under the curve (AUC) of 0.69 for PAPP-A, 0.76 for free beta-hCG and 0.75 for PNP, respectively (Fig. 7). In this model the combination of all three markers resulted in a similar AUC compared to the combination of PNP with free beta-hCG. Using a model including gestational age as a covariate, the AUC for PAPP-A was 0.87, for free beta-hCG 0.76 and for PNP 0.74, respectively (Fig. 8). However, combining all three markers and including gestational age as covariate was significantly better than all other models with an AUC of 0.93. The sensitivities and specificities of exemplary PNP concentration cut-off values are given in Table 3.

Results of logistic regression analysis for single and multimarker models of the MoM-values are presented in Table 4. ROC plot analysis for single markers revealed an area under the curve (AUC) of 0.82 for PAPP-A, 0.80 for free beta-hCG and 0.78 for PNP, respectively (Fig. 9). In this model the combination of all three markers was significantly better than all other models with an AUC of 0.91. The sensitivities and specificities of exemplary PNP-MoM cut-off values are given in Table 5.

The detection rates (DR) and false-positive rates (FPR) from including and excluding PNP, for risk cut-offs of 1 in 100 and 1 in 250 at time of screening, are given in Table 6. Including PNP in the risk calculation affects both, the DR and the FPR at fixed cut-offs of 1 in 100 and 1 in 250. There was a substantial reduction in FPR when PNP is added to first trimester biochemistry (ii) at a risk cut-off 1 in 250. Moreover, there was a substantial increase in the DR at risk cut-offs of 1 in 100 and 1 in 250 when PNP was added to the combined test (iv).

The detection rates for Down Syndrome at a fixed false-positive rate of 5% are given in Table 7 with 95% confidence intervals (CI) in brackets. There was a substantial increase in DR when PNP was added to first trimester biochemistry (ii) compared to biochemistry alone (i). Moreover, there was a substantial increase in DR when PNP is added to the combined test (iv) as compared to the combined test alone (iii). 3. Clinical assessment of PNP on patients with Trisomy 13 pregnancy

To test whether PNP is a useful marker for the diagnosis of Trisomy 13 (Pateau Syndrome), we evaluated PNP in serum samples from 246 women who underwent normal pregnancies and 30 samples from pregnant women carrying a fetus with Trisomy 13. PNP concentrations in this population of women with normal pregnancies ranged between 2.6 and 157.9 ng/niL with a median concentration of 21.7 ng/mL. In women with Trisomy 13 pregnancy, PNP serum concentrations were significantly elevated when compared to women with normal pregnancy (p = 0.014) with measuring values between 8.1 and 107.0 ng/mL and a median of 30.9 ng mL (Fig. 10). The respective values of PNP MoM (multiple of the median) are shown in Fig. 1 1. PNP MoM in women with normal pregnancy ranged between 0.12 and 7.1 with a median of 1.0, whereas woman with Trisomy 13 pregnancy MoM values were significantly elevated when compared to women with normal pregnany (p = 0.016) ranging between 0.37 and 4,9 with a median of 1.4.

Results of logistic regression analysis for single and multimarker models of the marker concentration levels (with and without gestational age as a covariate) are presented in Table 8. ROC plot analysis for single markers without inclusion of gestational age as a covariate revealed an area under the curve (AUC) of 0.93 for PAPP-A, 0.76 for free beta-hCG and 0.64 for PNP, respectively (Fig. 12). In this model the combination of all three markers resulted in a significantly higher AUC than for all other single markers and two-marker combinations. Using a model including gestational age as a covariate, the AUC for PAPP-A was 0.93, for free beta- hCG 0.77 and for PNP 0.67, respectively (Fig. 13). However, combining all three markers and including gestational age as covariate was significantly better than all other models with an AUC of 0.95. The sensitivities and specificities of exemplary PNP concentration cut-off values are given in Table 9.

Results of logistic regression analysis for single and multimarker models of the MoM-values are presented in Table 10. ROC plot analysis for single markers revealed an area under the curve (AUC) of 0.93 for PAPP-A, 0.77 for free beta-hCG and 0.64 for PNP, respectively (Fig. 14). In this model the combination of all three markers was significantly better than all other models with an AUC of 0.95. The sensitivities and specificities of exemplary PNP-MoM cut-off values are given in Table 11.

The detection rates (DR) and false-positive rates (FPR) of Trisomy 13 from including and excluding PNP, for risk cut-offs of 1 in 100 and 1 in 250 at time of screening, are given in Table 12. Including PNP in the risk calculation affects both, the DR and the FPR at fixed cut-offs of 1 in 100 and 1 in 250. There was a substantial reduction in FPR when PNP is added to first trimester biochemistry (ii) at a risk cut-off 1 in 250. Moreover, there was a substantial increase in the DR at risk cut-offs of 1 in 100 and 1 in 250 when PNP was added to the combined test (iv). The detection rates for Trisomy 13 at a fixed false-positive rate of 5% are given in Table 13 with 95% confidence intervals (CI) in brackets. There was a substantial increase in DR when PNP was added to first trimester biochemistry (ii) compared to biochemistry alone (i). 4. Clinical assessment of PNP on patients with Trisomy 18 pregnancy

To test whether PNP is a useful marker for the diagnosis of Trisomy 18 (Edward's Syndrome), we evaluated PNP in 30 serum samples from from pregnant women carrying a fetus with Trisomy 18 and compared them to PNP in 246 women who underwent normal pregnancies. In women with Trisomy 18 pregnancy, PNP serum concentrations were significantly elevated when compared to women with normal pregnancy (p - 0.012) with measuring values between 6.0 and 234.3 ng/mL and a median of 31.1 ng mL (Fig. 15). The respective values of PNP MoM (multiple of the median) are shown in Fig. 16. In women with Trisomy 18 pregnancy PNP MoM values were significantly elevated when compared to women with normal pregnany (p = 0.015) ranging between 0.27 and 10.7 with a median of 1.4.

Results of logistic regression analysis for single and multimarker models of the marker concentration levels (with and without gestational age as a covariate) are presented in Table 14. ROC plot analysis for single markers without inclusion of gestational age as a covariate revealed an area under the curve (AUC) of 0.988 for PAPP-A, 0.964 for free beta-hCG and 0.64 for PNP, respectively (Fig. 17). In this model the combination of all three markers resulted in a similar AUC compared to the combination of PAPP-A and free beta-hCG (0.994 and 0.994, respectively). Using a model including gestational age as a covariate, the AUC for PAPP-A was 0.987, for free beta-hCG 0.98 and for PNP 0.71, respectively (Fig. 18). Again, the combination of all three markers resulted in a similar AUC compared to the combination of PAPP-A and free beta-hCG (0.999 and 0.998, respectively). The sensitivities and specificities of exemplary PNP concentration cut-off values are given in Table 15.

Results of logistic regression analysis for single and multimarker models of the MoM-values are presented in Table 16. ROC plot analysis for single markers revealed an area under the curve (AUC) of 0.981 for PAPP-A, 0.976 for free beta-hCG and 0.64 for PNP, respectively (Fig. 19). In this model the combination of all three markers was similar to the combination of PAPP-A and free beta-hCG (AUC of 0.996 and 0.996, respectively). The sensitivities and specificities of exemplary PNP-MoM cut-off values are given in Table 17.

The detection rates (DR) and false-positive rates (FPR) of Trisomy 18 from including and excluding PNP, for risk cut-offs of 1 in 100 and 1 in 250 at time of screening, are given in Table 18.

The detection rates for Trisomy 18 at a fixed false-positive rate of 1% are given in Table 19 with 99% confidence intervals (CI) in brackets. There was a substantial increase in DR when PNP was added to first trimester biochemistry (ii) compared to biochemistry alone (i). Sequences

SEQ-ID No. 1 :

10 20 30 40 50 6£

MENGYTYEDY KNTAEWLLSH TKHRPQVAII CGSGLGGLTD KLTQAQIFDY GEIPNFPRST

70_ 80 90 100 110 120

VPGHAGRLVF GFLNGRACVM MQGRFHMYEG YPLWKVTFPV RVFHLLGVDT LWTNAAGGL

130 140 150 160 170 180

NPKFEVGDIM LIRDHINLPG FSGQNPLRGP NDERFGDRFP AMSDAYDRTM RQRALSTWKQ

190 200 210 220 230 240_

MGEQRELQEG TYVMVAGPSF ETVAECRVLQ KLGADAVGMS TVPEVIVARH CGLRVFGFSL

250 260 270 280

ITNKVIMDYE SLEKANHEEV LAAGKQAAQK LEQFVSILMA SIPLPDKAS

Tables

Table 1 : Weekly median MoMs for PNP, PAPP-A and free beta-hCG in women with pregnancy

Table 2: Logistic regression analysis of marker concentration values for diagnosis of DS

Table 3: Specificity and sensitivity values at different cut-off concentration levels for PNP to differentiate if a pregnant woman is carrying a fetus with fetal aneuploidy (DS)

PNP cut-off value (ng/ml) Specificity (in %) Sensitivity (in %)

10.1 37.1 89.7

14.7 51.7 76.9

18.6 62.3 71.8

24.6 74.8 56.4

36.9 84.8 53.9

53.7 94.7 43.6

68.8 99.3 35.9

86.8 100 25.6 Table 4: Logistic regression analysis of marker MoM-values for diagnosis of DS

Table 5: Specificity and sensitivity values at different cut-off PNP MoM values to differentiate if a pregnant woman is carrying a fetus with fetal aneuploidy (DS)

Table 6: Empirical results for the diagnosis of DS

Table 7: Detection rate of DS for a fixed false-positive rate (5%)

Table 8: Logistic regression analysis of marker concentration values for diagnosis of Trisomy 13

Table 9: Specificity and sensitivity values at different cut-off concentration levels for PNP to differentiate if a pregnant woman is carrying a fetus with Trisomy 13

Table 10: Logistic regression analysis of marker MoM-values for diagnosis of Trisomy 13

Marker i² AUC P

PNP 5.2 0.64 <0.05

Free beta-hCG 28.4 0.77 <0.001

PNP + free beta- 32.6 0.79 <0.001

hCG

PAPP-A 95.2 0.93 <0.001

PAPP-A + free 101.3 0.94 <0.001

beta-hCG

PNP + PAPP-A 105.8 0.95 <0.001

PNP 4- free beta- 112.3 0.95 <0.001

hCG + PAPP-A Table 11 : Specificity and sensitivity values at different cut-off PNP MoM values to differentiate pregnant woman is carrying a fetus with Trisomy 13

Table 12: Empirical results for the diagnosis of Trisomy 13

Table 13 : Detection rate of Trisomy 13 for a fixed false-positive rate (5%)

Table 14: Logistic regression analysis of marker concentration values for diagnosis of Trisomy

18

Model without covariate Model with covariate

gestational age gestational age

Marker x¹ AUC P x² AUC P p (Covariate)

PNP 6.6 0.64 <0.001 16.7 0.71 <0.01 <0.001

Free beta-hCG 1 15.0 0.96 <0.001 136.1 0.98 <0.001 <0.001

PNP + free beta- 1 15.1 0.96 O.001 136.33 0.98 <0.001 O.001 hCG

PAPP-A 138.0 0.988 <0.001 138.4 0.987 <0.001 >0.05

PAPP-A + free 161.5 0.994 <0.001 173.3 0.998 <0.001 <0.001 beta-hCG

PNP + PAPP-A 140.9 0.981 <0.001 141.1 0.981 <0.001 >0.05

PNP + free beta- 163.5 0.994 O.001 175.5 0.999 O.001 >0.05 hCG + PAPP-A Table 15: Specificity and sensitivity values at different cut-off concentration levels for PNP to differentiate if a pregnant woman is carrying a fetus withTrisomy 118

Table 16: Logistic regression analysis of marker MoM-values for diagnosis of Trisomy 18

Table 17: Specificity and sensitivity values at different cut-off PNP MoM values to differentiate if a pregnant woman is carrying a fetus with Trisomy 18

Table 18: Empirical results for the diagnosis of Trisomy 18

1 in 100 1 in 250

FP DR. FPR DR

(i) maternal age, PAPP-A & free b-HCG 1.6% 90.0% 1.6% 100.0%

(ii) maternal age, PAPP-A, free b-HCG & PNP 1.6% 90.0% 1.6% 96.7%

(iii) maternal age, PAPP-A, free b-HCG & NT 0.4% 100.0% 0.4% 100.0%

(iv) maternal age, PAPP-A, free b-HCG, PNP & NT 0.4% 100.0% 1.2% 100.0% able 19: Detection rate of Trisomy 18 for a fixed false-positive rate of 1%

Claims

A method to determine whether a pregnant woman has an increased risk of carrying a fetus with fetal aneuploidy comprising the steps of:

(i) providing a sample of a bodily fluid of pregnant women,

(ii) determining the level of PNP or fragments thereof and/or anti-PNP antibodies in said sample,

(iii) comparing said level with a reference level,

(iv) identifying whether the level is different from said reference level and evaluating whether the fetus has an increased risk of fetal-aneuploidy, if the level is different from the reference level,

(v) wherein an increased level of PNP or fragments thereof and/or anti-PNP-antibody in comparison to said reference level is an indication of an increased risk of fetal aneuploidy

wherein said fragments have a lengths of at least 6 amino acid residues.

A method of claim 1, wherein at least one further marker is determined selected from the group consisting of AFP, uE3, total hCG, alpha-hCG, beta-hCG, beta-core hCG, ITG, PGH, inhibin, inhibin A, PAPP-A, proMBP, complexes of PAPP-A with proMBP, proMBP complexes with angiotensinogen and/or complement factors and split products, ADAM12, cell-free fetal DNA or RNA, ultrasound markers, nuchal translucency, femur length, absence of nasal bone, fetal malformations, maternal age, maternal history or gestational age.

A method of claims 1 and 2, wherein the multiple of the median (MoM) or the degrees of extremeness (DoE) is calculated.

A method according to claims 1 to 3, calculating a first probability that the multiple of the median value is part of a Gaussian distribution of values found in unaffected pregnancies; calculating a second probability that the multiple of the median value is part of a Gaussian distribution of values found in pregnancies with fetal aneuploidy and calculating a likelihood ratio, wherein said likelihood ratio being said first probability divided by said second probability.

5. A method according to any of the claims 1 to 4, calculating a pregnant woman's prior risk of carrying a fetus having an increased risk of fetal aneuploidy and modifying said prior risk by the likelihood ratio to determine a combined risk.

6. A method according to claim 5, wherein a pregnant woman's prior risk of carrying a fetus having an increased risk of fetal aneuploidy is the age-related risk.

7. A method according to claims 1 to 6, wherein the determination of the level of PNP or fragments thereof and/or anti-PNP antibodies and/ or at least one further marker is carried out within the first to third trimester, more preferred within the first to second trimester (8th to 26th week of pregnancy), even more preferred within the first to early second trimester (8th to 20th week of pregnancy), even more preferred within the first trimester (8th to 14th week of pregnancy), mostly preferred within the early first trimester (8th to 10th week of pregnancy).

8. A method according to claims 1 to 7, further comprising adjusting any or all of the measured marker levels for one or more factors selected from the group of maternal race, maternal weight, smoking status, prior pregnancy adverse outcome, multiple birth and diabetic status.

9. A method according to claims 1 to 8, wherein marker measurements carried out within the first and second trimester are combined by integrated, sequential and/or contingent screening.

10. A method of any of claims 1 to 9, wherein fetal aneuploidy is selected from the group consisting of trisomy 21 (Down^'s syndrome), trisomy 18 (Edward's syndrome), trisomy 13 (Pateau syndrome), trisomy 12 (chronic lymphatic leukemia), trisomy 9 and trisomy 8 (Warkany Syndrome 2), monosomy X (Turner syndrome), trisomy X (Triple-X- Syndrome) and Klinefelter 's syndrome.

A method of any of claims 1 to 10, wherein said sample is a bodily fluid, in particular blood, serum, plasma, amniotic fluid or urine.

12. A method according to claims 1 to 11, further comprising comparing the quantitative estimate of the risk of fetal aneuploidy with a pre-determined cut-off to classify the pregnant woman as screen-positive or screen-negative based on the comparison.

13. A method of any of claims 1 to 12, wherein the patient is subjected to further testing selected from the group of high-resolution sonogram screening, amniocentesis and chorionic villus sampling when the pregnant woman was classified as screen-positve.

14. A method for determining a pregnant woman's risk of carrying a fetus with fetal aneuploidy in an analyzing system, the method, in an analyzing module of the analyzing system, comprising steps of:

receiving a measurement level of PNP or fragments thereof and / or anti-PNP antibodies,

receiving a measurement level of at least one further marker, and

determining a quantitative estimate of the risk of fetal aneuploidy by comparing the input levels of said markers with observed relative frequency distributions of marker levels in pregnancies affected with fetal aneuploidy and in unaffected pregnancies.

15. The method according to claim 14, wherein the step of determining the quantitative estimate comprises a step of deriving a likelihood ratio of fetal aneuploidy using a multivariate analysis of the measurement level of PNP or fragments thereof and / or anti- PNP antibodies and a measurement level of at least one further marker based on distribution parameters derived from a set of reference data.

16. The method according to claim 14 or 15, the method further comprising a step of re- expressing each input level of a marker as a multiple of the median level of the respective marker in unaffected pregnancies of the same gestational age as the fetus of the pregnant women, the re-expressed marker level being used in said determination of the risk of fetal aneuploidy.

17. The method according one of the claims 14 to 16, the method further comprising a step of re-expressing each input level of a marker as degrees of extremeness of the respective marker in unaffected pregnancies of the same gestational age as the fetus of the pregnant women, the re-expressed marker level being used in said determination of the risk of fetal aneuploidy.

18. The method according one of the claims 14 to 17, the method further comprising a step of comparing the quantitative estimate of risk with a predetermined cut-off level to classify the pregnant woman as screen-positive or screen-negative based on the comparison.

19. A computer program product for determining a pregnant woman's risk of carrying a fetus with fetal aneuploidy on a computer system, the computer product comprising:

receiving means for receiving a measurement level of PNP or fragments thereof and / or ¾nti-PNP antibodies and a measurement level of at least one further marker, and

determination means for determining a quantitative estimate of the risk of fetal aneuploidy by comparing the input levels of said markers with observed relative frequency distributions of marker levels in pregnancies affected with fetal aneuploidy and in unaffected pregnancies.

2Θ. A kit for determining the level of PNP or fragments thereof and/or anti-PNP antibodies wherein the kit comprises at least:

a first capture molecule that specifically binds a first epitope of SEQ-ID No. 1.

21. A kit according to claim 20, wherein the kit further comprises:

second capture molecule that specifically binds to a second epitope of SEQ-ID No. 1, that is different from said first epitope.

22. A kit for carrying out a method according to claim 20, wherein the kit comprises at least:

- a first capture molecule that specifically binds a first epitope of SEQ-ID No. 1.

23. A kit for carrying out a method according to claim 22, wherein the kit further comprises a second capture molecule that specifically binds to a second epitope of SEQ-ID No. 1, that is different from said first epitope.

24. A kit according to claims 20 to 23, wherein the capture molecule is an antibody.

25. A kit for determining the level of anti-PNP -antibody or fragments thereof is used, wherein the kit comprises at least: (ii) a first capture molecule that specifically binds a first epitope of said anti-PNP- antibody.

26. A kit for determining the level of anti-PNP-antibody or fragments thereof is used, wherein the kit comprises at least:

(iii) a first capture molecule that specifically binds a first epitope of said anti-PNP- antibody,

(iv) a second capture molecule that specifically binds to a second epitope of said anti- PNP-antibody, that is different from said first epitope.

27. A kit according to claims 25 to 26, wherein the capture molecule is an antigen and/or antibody.