CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of provisional application no. 61/359,568 filed on May 17, 2010.

BACKGROUND OF THE INVENTION

(1 ) Field of the Invention:

The present invention generally relates to markers for predicting fetal abnormalities. More specifically, the invention provides a marker useful for determining whether a pregnant woman is at elevated risk for carrying a fetus with a chromosomal abnormality.

(2) Description of the Related Art:

Prenatal screening for chromosomal abnormalities encompasses various techniques to gain knowledge about the karyotype of the fetus. The first serum screening test for aneuploidy was developed by Merkatz et al. (1984) when low concentrations of alpha-fetoprotein (AFP) in maternal serum was correlated with fetal DS. The use of serum AFP concentration combined with maternal age as a screening test for DS was brought into general practice over the subsequent years (New England Regional Genetics Group Prenatal Collaborative Study, 1989). Further serum markers have since been introduced into practice, including human chorionic gonadotropin (hCG) which is elevated (Bogart et al., 1987), unconjugated estriol (uE3), which is decreased (Canick et al., 1988) and inhibin A which is also elevated (Cuckle et al., 1996). See also Nicolaides et al., 2005; Wald et al., 2000; U.S. Pat. Nos. 4,874,693; 5,316,953; and 6,025,149; and International Patent Publication Nos. WO 90/08325; WO 94/03804; and WO 00/40428. At a 5% false positive rate, in combination with maternal age, maternal serum markers can detect approximately 70% to 80% of cases of fetal DS and by incorporating fetal ultrasound measurements, similar or slightly improved detection rates in the first trimester (ACOG Practice Bulletin 77). Due to the number of pregnant women affected, the issue of residual risk and false positive rates, and the serious ramifications with respect to reproductive health care, this issue of improved screening remains a priority.

Down syndrome, caused by the presence of an extra copy of chromosome 21 , is one of the most common chromosomal disorders and affects

approximately 1 in every 800 live births in the United States. It was first described by a British physician named John Langdon Down in 1866 and its cause was finally discovered in 1959 by Jerome Lejeune. Thereafter this disorder has been also referred to as Trisomy 21 (Harvey, 2004). The condition is often

characterized by moderate to severe physical and cognitive impairment. There is extremely wide disparity in the severity of these impairments, which are in most cases known to be associated with the extent of genetic abnormality (Harvey, 2004); (Roizen and Patterson, 2003). While Down syndrome makes up approximately 50% of chromosomal abnormalities detected at the time of amniocentesis, other chromosomal abnormalities with more severe sequelae may also be present and cause significant neonatal morbidity and mortality.

Definitive prenatal diagnosis of a chromosomal abnormality such as Down syndrome currently requires cytogenetic analysis of fetal cells obtained by using invasive procedures. The invasive procedures are associated with slight risk of fetal injury or miscarriage (ACOG Practice Bulletin 88). Therefore, screening tests for chromosomal abnormalities have been developed that are essentially non-invasive,systematic applications that provide an indication of the likelihood of having an aneuploid pregnancy and are a routine part of prenatal care. They are not definitive, but rather help identify high risk pregnancies so that diagnostic tests can then be offered. Screening tests can thus limit the number of pregnant women undergoing unnecessary invasive diagnostic procedures, which are associated with high costs and, importantly, risk of loss of a normal pregnancy due to a procedure-related miscarriage (Grant, 2005). Therefore, the addition of new markers for serum screening would allow for earlier detection and could be combined with other markers to enhance detection and specificity, reducing false positive and false negative results.

Previous microarray studies on aneuploid and normal placentae revealed differential expression of certain genes (Gross et al., 2002). Our research has demonstrated that Pregnancy-associated plasma protein-A2, also known as pregnancy associated plasma protein E and PLAC 3 is such a protein that is differentially expressed in aneuploid placentae such as Down syndrome.

Immunohistochemical analysis reveals that placental PAPP-A2 is more highly expressed in DS placentas than in normal controls (Figure 1 and Figure 2). It would be desirable to determine whether PAPP-A2 could be used as a bodily fluid (e.g. serum, urine) marker for chromosomal abnormalities, such as Down syndrome, either alone or in combination with other markers to enhance detection and reduce false positive and negative rates of Down syndrome. The present invention addresses that need by introducing a novel method of detection of increased risk of fetal chromosomal abnormalities based on assay of maternal PAPP-A2.

SUMMARY OF THE INVENTION

Accordingly, the inventors have discovered that pregnant women with elevated PAPP-A2 levels in bodily fluids have an elevated risk of carrying a fetus with a chromosomal abnormality.

Thus, the invention is directed to methods of determine whether a pregnant woman is at elevated risk for carrying a fetus with a chromosomal abnormality. The methods comprise

(a) obtaining a biological fluid sample from the woman;

(b) determining the concentration of PAPP-A2 in the biological fluid sample; and

(c) using the concentration determined in step (b) for determining whether the pregnant woman is at elevated risk for carrying a fetus with a chromosomal abnormality. The invention is also directed to kits or assays for determining whether a pregnant woman is at elevated risk for carrying a fetus with a

chromosomal abnormality. The assays or kits comprise a first detectable agent specific for PAPP-A2 or a component thereof and a second detectable agent specific for alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), pregnancy-associated plasma protein A (PAPP-A), estrogen, estriol, inhibin A or other markers incorporated to assess fetal risk for aneuploidy in any combination of one or more of these detectable agents. The assay method may include, but is not restricted to, immuno- based detection methods such as enzyme-linked immunosorbent assay (ELISA), Western blotting and radioimmunoassay (RIA).

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that PAPP-A2 concentration is generally elevated in bodily fluids of women carrying a fetus having Down syndrome and also altered in other chromosomal abnormalities. See Example. While PAPP-A2 has appeared elsewhere in the literature (Farr et al.,2000; Nishizawa et al., 2008; Overgaard et al., 2001 ; Page et al., 2001) it has not been previously established that PAPP-A2 concentration in bodily fluids correlates with any fetal abnormality. Based on this discovery, PAPP-A2 measurement in a bodily fluid of a pregnant woman can be used to determine whether the pregnant woman is at elevated risk for carrying a fetus having a chromosome abnormality. Thus, the invention is directed to methods of determining whether a pregnant woman is at elevated risk for carrying a fetus with a chromosomal abnormality. The methods comprise

(a) obtaining a biological fluid sample from the woman;

(b) determining the concentration of PAPP-A2 in the biological fluid sample; and

(c) using the concentration determined in step (b) for determining whether the pregnant woman is at elevated risk for carrying a fetus with a chromosomal abnormality.

As used herein, a chromosomal abnormality is a condition where there is a structural or numerical alteration from a normal chromosomal complement, where the alteration can be visualized using cytogenetic techniques such as karyotyping, fluorescent in situ hybridization, comparative genomic hybridization, bead array ratio analysis or sequencing technique. Chromosomal abnormalities include, but are not limited to aneuploidy (including but not limited to 47, XYY; trisomy 21 [Down syndrome]; trisomy 18 [Edwards syndrome];

trisomy 13 [Patau syndrome]; 47, XXY [Klinefelter's syndrome]; monosomy 18; 45, X [Turner syndrome]; and 47, XXX), triploidy, tetraploidy, inversions

(including paracentric and pericentric inversions), translocations (including reciprocal and Robertsonian translocations), deletions (including terminal [telomeric] and interstitial deletions, cri du chat syndrome [5p-syndrome], and Wolf-Hirschhom syndrome [4p-deletion]), insertions, duplications, ring

chromosomes, isochromosomes, or mosaics having cells comprising any of the above abnormalities.

The determination step ( c ) of these methods can utilize any means of determining whether the concentration of PAPP-A2 in the sample is elevated. Preferably, that step involves comparing the concentration determined in step (b) with control concentration(s) from pregnant women at are not carrying fetuses with chromosomal abnormality. Here, if the PAPP-A2 concentration in the biological fluid sample is greater than control concentration, then the pregnant woman has an elevated risk of carrying a fetus with Down syndrome or other chromosomal abnormality. Additionally, if the PAPP-A2 concentration is not altered, then the pregnant woman does not have an elevated risk of carrying a fetus with a chromosomal abnormality.

A skilled artisan can design and execute the collection of the control fluid and measurement of the PAPP-A2 concentration in that control fluid without undue experimentation. Preferably, the control fluid is matched as much as possible with the sample fluid, e.g. in fluid type such as gestational age. The control fluids preferably should come from multiple women such that a

measurement of variation of PAPP-A2 concentration among control fluids can be ascertained, using statistical tools.

As used herein, "greater than" or "less than" the PAPP-A2 control concentration means a difference in concentration that is different enough from the control concentration(s) to achieve a desired detection level and/or false positive rate. These values can be determined by the skilled artisan without undue experimentation using well-known statistical tools. In some cases these statistical goals can be achieved through multiple sampling, e.g. at weekly intervals or retesting after achieving a significant difference from the control value.

Preferably, these methods are used to evaluate whether the woman is carrying a fetus with a chromosomal abnormality, such as Down syndrome.

It is expected that the methods of the present invention can utilize any biological fluid including the not limited to saliva, plasma, bile, lymph, mucus, or preferably urine or peripheral blood serum or matched samples of each.

Peripheral blood serum may be most preferred since other tests for aneuploidies most often utilize maternal serum, so the same serum sample can conveniently be used in the method of the present invention and in other screening tests for chromosomal abnormalities.

If the invention method shows an elevated risk for a fetus with a chromosomal abnormality, the method preferably further comprises offering the woman further testing for diagnosis. The further testing can include any test that can more definitively determine whether the pregnant woman is carrying an aneuploid fetus. Such tests can include but are not limited to sonography (fetal ultrasound) or amniocentesis or chorionic villus sampling.

These methods can also include tests for other known markers of chromosome abnormalities, such that the combination can achieve a higher detection level and/or a lower false positive rate than either test alone.

Nonlimiting examples of such tests include determining the concentration of alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), unconjugated estriol, (uE3), pregnancy-associated plasma protein A (PAPP-A), estriol, inhibin, fetal cells/nucleic acid extracted from maternal biological fluid samples.

Preferably, the above are determined along with PAPP-A2.

The methods of the present invention can be used with a woman in the first trimester or in the second trimester of pregnancy (see Example).

The concentration of PAPP-A2 can be determined by measuring this protein with an assay designed to detect PAPP-A2 or by a component thereof.

The invention is also directed to kits for determining whether a pregnant woman is at elevated risk for carrying a fetus with a chromosome abnormality. The kits may include, but is not restricted to, immuno-based detection methods such as enzyme-linked immunosorbent assay (ELISA), Western blotting and radioimmunoassay (RIA). The kits or asays comprise

A first detectable agent specific for PAPP-A2 or a component thereof and

A second detectable agent specific for alpha. -fetoprotein (AFP), human chorionic gonadotropin (hCG), unconjugated estriol (uE3), pregnancy- associated plasma protein A (PAPP-A), estriol, inhibin A, maternal cells/DNA in maternal biological fluid specimen and any combination of one or more of these detectable agents. The kits of the present invention are not limited to any particular detectable agent specific for PAPP-A2, AFP, hCG, uE3, PAPP-A, estriol, inhibin A. Nonlimiting examples of such useful agents include aptamers and naturally occurring proteins that bind or otherwise interact with the above. Preferably, the agent comprises an antibody binding site that is specific for PAPP-A2, AFP, hCG, uE3, PAPP-A, estriol, inhibin A or other such marker used for the purpose of fetal chromosomal screening. Since proteins containing specific antibody binding sites can be routinely generated by a number of methods known in the art, it is thus preferred that all of the detectable agents comprise an antibody binding site, most preferably antibodies.

The assays or kits are also not limited to any particular method of detecting the detectable agents. Such methods can include, for example, induction of a visible change to living cells in the assay (e.g., induction or suppression of apoptosis), utilization of a radioactive, enzymatic or fluorescent label on the detectable agent (e.g. antibody), or on a second agent that specifically binds to the first agent (e.g. second antibody), etc. Myraid such methods are known in the art.

The first detectable agent is preferably an antibody preparation that is specific for PAPP-A2. the antibody preparation can be any preparation comprising an antibody binding site specific for PAPP-A2, e.g. monoclonal antibody, polyclonal antibody, recombinant antibody or other proteins comprising an antibody binding site including single chain antibodies. The first detectable agent can also be specific for a component of PAPP-A2. Preferred embodiments of the invention are described in the following example. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the example, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the example.

Example. Correlation of PAPP-A2 with Down syndrome and other chromosomal abnormalities

Experimental design:

PAPP-A2 is a polypeptide belonging to the metazincin family. It is putatively classified as a metalloprotease due to the conserved amino acid stretches it shares with other members of this family of proteases. PAPP-A2 is an active enzyme expressed abundantly in human placenta and in non pregnant mammary gland (Overgaard et al., 2001). The enzyme is also expressed to a lower extent in various other tissues, including the kidney, fetal brain and pancreas (Page et al., 2001). It contains 1542 amino acid. Using

immunohistochemical technique, we were able to demonstrate differential protein expression (Figure 1 and Figure 2). To determine whether PAPP-A2 is a useful marker for Down syndrome and other fetal chromosomal abnormalities, we applied two approaches: Western blot and ELISA technique. Western blotting analysis. Protein concentrations of the serum samples were determined by Bradford assay with bovine serum albumin as protein standard. SDS sample buffer 4X (Invitrogen) was added to the samples and the samples were denatured at 95°C for 4 minutes. Gel electrophoresis was carried out and the proteins were separated using NuPAGE® Novex 3-8% Tris-Acetate Gel 1.5 mm, 10 welled (Invitrogen, Carlsbad, CA). A positive control, pure PAPP-E protein (R&D systems), was used to demonstrate that the protocol is efficient and that the antibody recognizes the target protein. The proteins were then

transferred to polyvinylidene difluoride membranes (Invitrogen). The membranes were wetted with TBS-T (Tris-buffered saline with 0.1 % Tween-20, pH 7.8) for 10 minutes, and were blocked with Western blocker solution (Sigma) for 120 minutes. The membranes were then hybridized with primary antibodies diluted in the blocker solution overnight at 4° C. Anti-PAPP-A2 primary antibody (R&D systems) was diluted to 0.66 pg/ ml (1 :1500 dilution).The membranes were washed with TBS-T several times and incubated with secondary antibody, anti- rabbit IgG, horseradish peroxidase-linked whole antibody (GE Healthcare, Buckinghamshire UK), at a 1 :2500 dilution in Western blocker solution at room temperature for 120 minutes. The membranes were washed again with TBS-T three times for 10 minutes and treated with the ECL Plus Western blotting detection system (GE Healthcare) and chemifluorescence was detected by exposure to x-ray film (Kodak). Densitometric quantification of the Western blot signal intensity of the bands was performed by densitometric analysis program image J (from NIH). The density values of albumin were used for normalization. The membranes were washed again with TBS-T three times for 10 minutes and were stripped by incubating in stripping buffer at 50° C for 30 minutes .The Western blotting was carried out using albumin primary antibody (Sigma, 1 :1000 dilution) according to the procedure described earlier.

Results: The PAPP-A2 band was observed at about 250 kDa. Serum levels of PAPP-A2 were significantly higher in aneuploid pregnancies compared to uncomplicated pregnancies (p<0.05) in the Western blotting experiments (Figure 3). In DS pregnancies, median maternal serum PAPP-A2 levels were

significantly elevated to 2.69 multiples of the median (MoM) (p<0.05) when compared with uncomplicated pregnancies (Figure 3).

Enzyme-linked immunsorbent assay (ELISA): PAPP-A2 concentrations were measured by a sandwich enzyme-linked immunosorbent assay (ELISA) using a kit (USCN Life Science Inc) and following the manufacturer's protocol.

Statistics: Mathematical algorithms to determine Down syndrome risk are calculated by determining the medians for the normal population and the down syndrome population. The greater the difference in the medians and the smaller the standard deviations for the curves, the more useful the marker. Multiples of the median are used in order to standardize the findings.

Results: A total of 101 serum samples were assayed, including 63 normal controls and 38 samples from DS pregnancies which were matched for gestational age. Samples were also assayed for first trimester markers and second trimester markers depending on gestational age. Getational ages included both first and second trimester, from 11 to 19 weeks. Multiples of the median levels (MoM) of PAPP-A2 in the DS samples when compared to matched gestational age normal controls range from 1.29 to 3.72. For all gestational ages tested, >90% detection was achieved with a 5% false positive rate. Increases in MoM was present at all gestational ages and were highly significant in DS samples as compared to normal controls at 15 weeks' (p<0.0001), 16 weeks' (p<0.0001) and 17 weeks' (p=0.0002) gestation. PAPP-A2 levels showed only partial correlation with other markers thus indicating benefit in combination with other markers. PAPP-A2 was also elevated in cases of aneuploidy distinct from T21 , with MoM's up to 2.94.

BRIEF DESCRIPTION OF FIGURES

Figure 1. Immunohistochemical analysis reveals that placental PAPP-A2 is more highly expressed in DS placentas than in normal controls.

Figure 2. Analysis of immunohistochemical staining by three blinded observers reveals significant difference in PAPP-A2 immunostaining between DS and normal controls (p<0.05). Figure 3. Serum levels of PAPP-A2 were significantly higher in aneuploid pregnancies compared to uncomplicated pregnancies (p<0.05) in the Western blotting experiments.

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U.S. Pat. No. 5,324,667

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International Patent Publication No. WO 90/08325 • International Patent Publication No. WO 94/03804

• International Patent Publication No. WO 00/40428

In view of the above, it will be seen that the several advantages of the invention are achieved and other advantages attained.

As various changes could be made in the above methods and

compositions without departing from the cope of the invention, it is intended that all matter contained in the above description and shown in the figures shall be interpreted as illustrative and not in a limiting sense.

All references cited in this specification are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.