HK1154407B - Markers for prenatal diagnosis and monitoring - Google Patents

Description

Markers for prenatal diagnosis and monitoring

Cross reference to related applications

This application claims priority from U.S. provisional application No. 60/663,293, filed on 18/3/2005, the contents of which are incorporated herein by reference in their entirety.

Background

Cells isolated from a fetus may be used for routine prenatal diagnosis by methods such as Chorionic Villus Sampling (CVS) or amniocentesis. However, despite extreme care, these conventional methods remain invasive and present a significant risk to the mother and fetus (Tabor et al, Lancet 1: 1287-1293, 1986).

Alternative methods to these invasive methods have been developed, for example, based on the finding that several foetal cells can be found in the maternal circulation, foetal abnormalities can be determined (Johansen et al, Presat Diagn.15: 921 931, 1995), more importantly, non-cellular circulating foetal DNA can be determined in maternal plasma and serum (Lo et al, Lancet 350: 485 487, 1997). It has now been shown that the amount of fetal DNA in maternal blood is sufficient for genetic analysis without the need for complex processing of plasma or serum, compared to the necessary steps for isolating and enriching fetal cells in maternal circulation. Fetal rhesus D (RhD) genotyping (Lo et al, N.Engl. J.Med.339: 1734-.

Furthermore, abnormal amounts of fetal DNA in maternal plasma/serum have been reported in preeclampsia (Lo et al, Clin. chem.45: 184-. The determination of fetal nucleic acid in maternal blood for prenatal genetic analysis has also been disclosed in U.S. patent No. 6,258,540.

When analyzing fetal DNA, researchers typically use as fetal-specific markers Y-chromosomal markers that are only present in male fetuses. This method limits the application of its technology to 50% of pregnant women, who are pregnant with a male fetus. Moreover, the use of other genetic polymorphisms also increases the complexity of fetal DNA-based analysis. Fetal RNA found in maternal plasma offers a possible new approach to overcome these limitations (Poon et al, Clin. chem.46: 1832-.

Recently, U.S. patent application No. 09/876,005 discloses a non-invasive technique based on the determination of fetal/placental RNA in maternal blood. In addition, U.S. patent application No. 10/759,783 discloses certain placenta-expressed mRNA markers (e.g., human chorionic gonadotropin beta subunit and human corticotropin releasing hormone) that can be used to assay for pregnancy and pregnancy related conditions such as preeclampsia, fetal chromosomal aneuploidy, and preterm labor. Other RNA types of various placental origins have been assayed in maternal blood, see, e.g., Oudejans et al, Clin chem.2003, 49 (9): 1445-: 1413-1414. The present invention also discloses other RNA types derived from the fetus/placenta, as shown in tables 1 to 6, which are found in maternal blood and are useful as markers for detecting pregnancy, or fetal genotyping, or diagnosing, monitoring and predicting preeclampsia and fetal chromosomal aneuploidy such as trisomy 18 and trisomy 21. Thus, the present invention also provides other tools for non-invasive prenatal diagnosis and alternative methods of pregnancy detection.

Summary of The Invention

In a first aspect, the present invention relates to a method of diagnosing, monitoring or predicting preeclampsia in a pregnant woman. The method comprises the following steps: first, the content of one or more RNA types in the biological sample of the pregnant woman is quantitatively determined. The RNA type is independently selected from the group consisting of RNA types derived from genetic loci comprising IGFBP3, ABP1, FN1, SLC21a2, KIAA0992, TIMP3, LPL, INHBA, LEP, ADAM12, PAPPA2, and SIGLEC6, and the biological sample is blood, washes of the reproductive tract, amniotic fluid, urine, saliva, or chorionic villi. Secondly, the amount of said RNA type in the first step is compared to a standard control for the amount of said RNA type in a corresponding sample from a normal non-preeclamptic pregnant woman. An increase or decrease in the amount of the RNA type relative to the standard control indicates pre-eclampsia or a high susceptibility to developing pre-eclampsia.

In some embodiments, the RNA type is derived from ADAM12, PAPPA2, FN1, INHBA, LEP, or SIGLEC6, and an increase in the amount of the RNA type as compared to the standard control indicates preeclampsia or a high susceptibility to developing preeclampsia. In other embodiments, the RNA type is derived from PAPPA, and the amount of the RNA type is reduced from the standard control by a factor that indicates or is highly susceptible to developing preeclampsia.

In some embodiments, the first step comprises using reverse transcription polymerase chain reaction (RT-PCR). Optionally, the first step may also include the use of mass spectrometry after RT-PCR. In other embodiments, the first step comprises using a polynucleotide hybridization method, or using a primer extension reaction.

In some embodiments, the pregnant woman being detected is in the first trimester of pregnancy. In other embodiments, the pregnant woman is in the middle or last trimester of pregnancy.

In some embodiments, the blood is fractionated and the plasma fraction is analyzed. In other embodiments, the blood is fractionated and the serum fraction is analyzed. In some embodiments, the RNA content is increased more than 2-fold over a standard control. In other embodiments, the RNA content is reduced by more than 50% over a standard control.

The invention also provides kits for diagnosing, monitoring or predicting preeclampsia in a pregnant woman. The kit comprises: (i) PCR primers for quantifying the amount of one or more RNA species in a biological sample from said pregnant woman, wherein said RNA species are independently selected from the group consisting of RNA species derived from a genetic locus comprising IGFBP3, ABP1, FN1, SLC21a2, KIAA0992, TIMP3, LPL, INHBA, LEP, ADAM12, PAPPA2 and SIGLEC6, wherein said biological sample is blood, a wash of the reproductive tract, amniotic fluid, urine, saliva or chorionic villi; and (ii) a standard control representative of the amount of said RNA type in a corresponding sample from a normal non-preeclamptic pregnant woman.

In a second aspect, the present invention relates to a method for determining the presence of trisomy 18 in a fetus of a pregnant woman, said method comprising the steps of: first, the amount of an RNA type derived from RPL17 in a biological sample of the pregnant woman is quantified. The biological sample is blood, washing of genital tract, amniotic fluid, urine, saliva or chorionic villus. Secondly, the amount of said RNA type in the first step is compared with a standard control for the amount of said RNA type in a corresponding sample of a pregnant woman with a normal chromosomal fetus. Deviation of the RNA type content from the standard control indicates that the pregnant fetus has a high susceptibility to trisomy 18.

In some embodiments, an increase in the RPL17RNA content relative to a standard control indicates a high susceptibility of chromosome 18 of the pregnant infant to trisomy; in other cases, a decrease in the amount of RPL17RNA relative to the standard control may also indicate that the fetus has a high susceptibility to trisomy 18.

In some embodiments, the first step comprises using reverse transcription polymerase chain reaction (RT-PCR). Optionally, the first step may also include the use of mass spectrometry after RT-PCR. In other embodiments, the first step comprises using a polynucleotide hybridization method, or using a primer extension reaction.

In some embodiments, the pregnant woman being detected is in the first trimester of pregnancy. In other embodiments, the pregnant woman is in the middle or last trimester of pregnancy.

In some embodiments, the blood is fractionated and the plasma fraction is analyzed. In other embodiments, the blood is fractionated and the serum fraction is analyzed. In some embodiments, the amount of RNA is increased more than 2-fold over a standard control. In other embodiments, the amount of RNA is reduced by more than 50% from a standard control.

Also provided are kits for determining the presence of trisomy 18 in a fetus of a pregnant woman. The kit comprises: (i) PCR primers for quantifying the amount of an RNA type in a biological sample of said pregnant woman, wherein said RNA type is derived from RPL17, and wherein said biological sample is blood, washings of the genital tract, amniotic fluid, urine, saliva or chorionic villi; and (ii) a standard control representative of the amount of said RNA type in a corresponding sample from a pregnant woman carrying a normal chromosomal fetus.

In a third aspect, the present invention relates to a method for determining the presence of trisomy 21 in a fetus of a pregnant woman. The method comprises the following steps: first, quantitatively determining the amount of an RNA type in a biological sample of said pregnant woman, wherein said RNA type is independently selected from the group of RNA types derived from a genetic locus comprising COL6a1, COL6a2, SOD1, APP, BTG3, ATP5J, ADAMTS1, BACE2, DSCR5, ITSN1, PLAC4, ATP5O, LOC90625, EFEMP1 and TFRC, wherein said biological sample is blood, a wash of the reproductive tract, amniotic fluid, urine, saliva or chorionic villi. Secondly, the RNA type content in the first step is compared with a standard control for the RNA type content in a corresponding sample of a pregnant woman with a normal chromosomal fetus. An increase or decrease in the amount of the RNA type relative to the standard control indicates that the pregnant fetus has a high susceptibility to trisomy 21.

In some embodiments, the RNA type is derived from ADAMTS1, APP, ATP5O, EFEMP1, or TFRC, wherein an increase in the RNA type content as compared to the standard control indicates that the pregnant fetus has a high susceptibility to trisomy 21.

In some embodiments, the first step comprises using reverse transcription polymerase chain reaction (RT-PCR). Optionally, the first step may also include the use of mass spectrometry after RT-PCR. In other embodiments, the first step comprises using a polynucleotide hybridization method, or using a primer extension reaction.

In some embodiments, the pregnant woman being detected is in the first trimester of pregnancy. In other embodiments, the pregnant woman is in the middle or last trimester of pregnancy.

In some embodiments, the blood is fractionated and the plasma fraction is analyzed. In other embodiments, the blood is fractionated and the serum fraction is analyzed. In some embodiments, the RNA content is increased more than 2-fold over a standard control. In other embodiments, the RNA content is reduced by more than 50% over a standard control.

Also provided are kits for determining the presence of trisomy 21 in a fetus of a pregnant woman. The kit comprises: (i) PCR primers for quantitative determination of the amount of an RNA type in a biological sample of said pregnant woman, wherein said RNA type is independently selected from the group of RNA types derived from a genetic locus comprising COL6a1, COL6a2, SOD1, APP, BTG3, ATP5J, ADAMTS1, BACE2, DSCR5, ITSN1, PLAC4, ATP5O, LOC90625, EFEMP1 and TFRC, and wherein said biological sample is blood, a wash of the reproductive tract, amniotic fluid, urine, saliva or chorionic villi; and (ii) a standard control representative of the amount of said RNA type in a corresponding sample from a pregnant woman carrying a normal chromosomal fetus.

In a fourth aspect, the present invention relates to a method of determining pregnancy in a woman. The method comprises the following steps: first, the amount of RNA species in the biological sample of the woman is quantified. The RNA type is independently selected from the group consisting of RNA types derived from a genetic locus comprising COL6a1, COL6a2, SOD1, ATP5O, ADAMTS1, DSCR5, and PLAC4, wherein the biological sample is blood, a wash of the reproductive tract, amniotic fluid, urine, saliva, or chorionic villi. Secondly, the amount of said RNA type in the first step is compared with a standard control for the amount of said RNA type in a corresponding sample from a normal non-pregnant woman. An increase or decrease in the amount of the RNA type compared to the standard control is indicative of pregnancy.

In some embodiments, the RNA type is derived from COL6a1, COL6a2, ATP5O, or PLAC4, an increase in the amount of the RNA type compared to the standard control is indicative of pregnancy.

In some embodiments, the first step comprises using reverse transcription polymerase chain reaction (RT-PCR). Optionally, the first step may also include the use of mass spectrometry after RT-PCR. In other embodiments, the first step comprises using a polynucleotide hybridization method, or using a primer extension reaction.

In some embodiments, the detected woman is in the first trimester of pregnancy. In other embodiments, the woman is in the middle or last trimester of pregnancy.

In some embodiments, the blood is fractionated and the plasma fraction is analyzed. In other embodiments, the blood is fractionated and the serum fraction is analyzed. In some embodiments, the amount of RNA is increased more than 2-fold over a standard control. In other embodiments, the amount of RNA is reduced by more than 50% from a standard control.

Kits for determining pregnancy in a woman are also provided. The kit comprises: (i) PCR primers for quantitative determination of the amount of RNA types in a biological sample from said woman, wherein said RNA types are independently selected from the group of RNA types derived from the genetic locus comprising COL6a1, COL6a2, SOD1, ATP5O, ADAMTS1, DSCR5 and PLAC4, and wherein said biological sample is blood, washes of the genital tract, amniotic fluid, urine, saliva or chorionic villi; and (ii) a standard control representative of the amount of said RNA type in a corresponding sample from a normal non-pregnant woman.

Brief description of the drawings

FIG. 1 is a graph showing a comparison of RNA transcript levels in placental tissue in chromosome 21 trisomy of the first trimester of pregnancy and in control pregnancies. (A) Represents ADAMTS1 mRNA. (B) Represents APP mRNA. Each ● represents a subject.

FIG. 2 shows the relative concentrations of placental expressed transcripts in the maternal buffy coat. The horizontal lines inside the boxes represent median values. The boxes represent the interval 25% -75%. The whisker line (whisker) represents the interval of 10% to 90%. The filled circles represent data outside the 10% -90% interval.

FIG. 3 shows placental mRNA clearance from maternal plasma after delivery. (A) And (B) concentrations of COL6a 1mRNA and COL6A2mRNA in maternal plasma before and 24 hours after delivery, respectively. Each line represents paired plasma samples from the same subject.

FIG. 4 is a graph showing a comparison of RNA transcript levels in placental tissue in chromosome 21 trisomy of the first trimester of pregnancy and control pregnancies. (A) Represents EFEMP1 mRNA. (B) Represents TFRC mRNA. (C) Represents ATP5O mRNA. Each ● represents a subject. (D) Indicating clearance of ATP5O mRNA in maternal plasma 24 hours after delivery. Each line represents paired plasma samples from the same subject.

FIG. 5 is a graph showing a comparison of placental tissue RNA transcript levels in preeclampsia in the third trimester (PET) versus control pregnancies. (A) Represents Leptin (Leptin, LEP) mRNA. (B) Represents SIGLEC6 mRNA. The horizontal lines inside the boxes represent median values. The boxes represent the interval 25% -75%. The whiskers represent the interval 10% -90%. The filled circles represent data outside the interval of 10% -90%.

FIG. 6 shows a comparison of Leptin (LEP) mRNA and INHBA mRNA concentrations in maternal plasma in preeclampsia versus control pregnancies, where (A) represents Leptin (LEP) mRNA. (B) Represents INHBA mRNA. The horizontal lines inside the boxes represent median values. The boxes represent the interval 25% -75%. The whiskers represent the interval 10% -90%. The filled circles represent data outside the interval of 10% -90%.

FIG. 7 is a block diagram showing plasma concentrations of PLAC4mRNA in women in primordial, first trimester and third trimester. The horizontal lines inside the boxes represent median values. The boxes represent the interval 25% -75%. The whiskers represent the interval 10% -90%. The filled circles represent data outside the interval of 10% -90%.

FIG. 8 shows the clearance of PLAC4mRNA from maternal plasma after delivery. Each line represents paired plasma samples from the same subject.

FIG. 9 is a graph showing a comparison of placental tissue RNA transcript levels in preeclampsia in the third trimester (PET) versus control (normal) pregnancies. (A) Representing LEP mRNA. (B) Represents ADAM12 mRNA. (C) Represents PAPPA mRNA. (D) Represents PAPPA2 mRNA. (E) Represents INHBAmRNA. (F) Represents FN1 mRNA. The horizontal lines inside the boxes represent median values. The boxes represent the interval 25% -75%. The whiskers represent the interval 10% -90%. The filled circles represent data outside the interval of 10% -90%.

Definition of

In the present specification, the term "RNA type derived from a genetic locus" refers to a polymer of ribonucleotides having a sequence corresponding to at least a part of a preselected position in the human genome. In the present application, an "RNA species" may or may not encode a protein product, whose sequence may comprise non-coding sequences or only a portion of an open reading frame.

In this specification, the terms "fetus", "derived from the placenta" or "placental expressed" refer to the source of certain RNA types that can be detected in a biological sample, such as blood, of a pregnant woman. In other words, the fetal RNA type is transcribed from a fetal DNA sequence. Furthermore, the types of RNA derived from or expressed by the placenta are found in the placenta and are transcribed from fetal DNA sequences.

In this specification, the term "washings of the genital tract" refers to any liquid or solution collected after rinsing or washing the genital tract of a pregnant woman or a woman testing for pregnancy.

In the present specification, the term "preeclampsia" refers to a disease state that occurs in pregnancy, the main symptoms of which are various hypertensions usually accompanied by proteinuria and edema (swelling). Preeclampsia, also sometimes referred to as toxemia of pregnancy, is also related to a more severe condition known as "eclampsia", which is preeclampsia accompanied by seizures. These states usually develop in the second half of pregnancy (after 20 weeks), although it is also possible to develop shortly after birth or before 20 weeks of pregnancy.

In the present specification, the term "primer extension reaction" refers to any polymerization process that extends a predetermined polynucleotide sequence that is at least partially complementary to a template sequence, under appropriate conditions, mediated by the action of a nucleotide polymerase, such as a DNA polymerase.

In the present specification, the term "chromosomal aneuploidy" refers to a chromosomal abnormality in which the number of chromosomes is not an integral multiple of the normal haploid number: it is often the case that there is an extra chromosome or one of the chromosomes is missing. The most common case of a chromosomal aneuploidy is a trisomy, which has one extra chromosome. For example, trisomy 18 is a chromosomal abnormality in which chromosome 18 is found in cells, while trisomy 21 is found in cells in which trisomy 21 is present.

In contrast to aneuploidy, "normal chromosomes" describes a situation where the number of chromosomes is an integer multiple of the number of haploids, e.g., twice the number of chromosomes in a diploid, and each chromosome has the same number (unless in a sex chromosome, e.g., in a male, two different sex chromosomes, X and Y are present in a single copy, respectively).

In this specification, the term "blood" refers to a blood sample or product from a pregnant woman or a woman tested for pregnancy. The term also includes whole blood, or any blood component of maternal or fetal origin, including platelets, at varying concentrations, even free of hematopoietic cells or any other type of cells or cell residues. "blood" includes plasma and serum. Blood samples that are substantially free of cells are also referred to as "cell-free," in that they are generally free of platelets.

In this specification, the term "normal" describes a reference to an indicator, such as the level of RNA derived from the fetus/placenta detected in the maternal blood, that is absent of preeclampsia, or a pregnant woman carrying a normal fetus with chromosomes, that is a value typical of a randomly selected group of women who are absent of preeclampsia, or who carry normal fetuses with chromosomes. The selected group should comprise a sufficient number of women such that the average level of fetal/placental derived RNA transcribed from a genetic locus (which may or may not encode a particular fetal protein) reflects with reasonable accuracy the level of said RNA in the general population of healthy pregnant women carrying healthy fetuses. In addition, the selected group of women should have a gestational age similar to that of the woman whose blood is assayed to account for preeclampsia or fetal chromosomal aneuploidy, e.g., trisomy 18 and trisomy 21. The preferred gestational age at which the present invention is practiced will vary depending upon the condition to be screened. For example, preeclampsia screening for a pregnant woman may preferably be performed during the middle third month of pregnancy, while preferably screening and diagnosis of fetal chromosomal aneuploidies should be performed as early as possible. Furthermore, given that certain markers may be easier to measure at some stage of pregnancy than at other stages, it is preferred that the gestational age at which the determination is made be dependent on the RNA marker used in the assay.

In the present specification, the term "normal" is similarly used to refer to the amount of a particular RNA species, which is a typical amount measured in the blood of a randomly selected group of healthy, non-pregnant women.

In this specification IGFBP3, ABP1, FN1, SLC21a2, KIAA0992, TIMP3, LPL, INHBA, LEP, SIGLEC6, RPL17, COL6a1, COL6a2, SOD1, APP, BTG3, ATP5J, ADAMTS1, BACE2, DSCR5, ITSN1, PLAC4, LOC90625, ATP5O, EFEMP1 and TFRC refer to genes or proposed open reading frames (including variants and mutants) and their polynucleotide transcripts, such as the sequences corresponding to GenBank accession numbers provided in tables 2, 4 and 6. In some places herein, these terms may also be used to indicate polypeptides encoded by these genes or open reading frames.

In the present specification, the term "standard control" refers to a sample suitable for use in the method of the invention for determining the level of RNA transcript, such as COL6A1, COL6A2, APP, ATP5O or LEP. These samples contain known amounts of fetal/placental derived RNA species that can be fairly closely reflected in the average levels of these RNA species in normal pregnant women. Similarly, a "standard control" may also be from a normal healthy, non-pregnant woman.

In the present specification, the term "the amount of said RNA type is increased or decreased compared to said standard control" refers to a positive or negative change in the amount relative to said standard control. The increase is preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 10-fold. Similarly, the reduction is preferably at least 50%, more preferably at least 80%, most preferably at least 90%.

In this specification, the term "polynucleotide hybridization method" refers to a method of determining the presence and/or amount of a certain polynucleotide based on its ability to form Watson-Crick base pairs with a polynucleotide probe of known sequence under appropriate hybridization conditions. Examples of such hybridization methods include Southern hybridization and Northern hybridization.

In the present specification, the term "PCR primer" refers to an oligonucleotide that can be used in a polymerase chain reaction to amplify a nucleotide sequence derived from an RNA transcript of a genetic locus such as COL6a1, COL6a2, APP, ATP5O or LEP. At least one PCR primer that amplifies an RNA sequence derived from the locus named above is sequence specific for the locus.

Detailed description of the invention

Summary of the invention

The present invention provides for the first time methods and kits for diagnosing, monitoring or predicting preeclampsia in a pregnant woman, trisomy 18 and trisomy 21 in a fetus, and pregnancy in a woman by analyzing the levels in the blood of the woman of one or more fetal/placental derived RNA types, i.e., having the sequences set forth in tables 1-6.

The amount of fetal/placental derived RNA transcripts in these maternal blood samples may preferably be determined quantitatively after completion of the amplification process, e.g. reverse transcriptase polymerase chain reaction (RT-PCR), as described herein. The amount of one or more of these RNA types is then compared to a standard control with the same type of RNA level that is typical for normal pregnant women at a similar gestational age but without these pregnancy-associated disorders. An increase or decrease in the level of the RNA is indicative of the presence of the disorder or a heightened susceptibility to develop the disorder. Thus, the present invention provides a novel approach to diagnose preeclampsia, as well as fetal chromosomal aneuploidy, such as fetal trisomy 18 and 21, that is non-invasive and independent of gender and polymorphism.

The present invention can also be used to determine pregnancy by comparing the levels of one or more of said RNA types transcribed from these genetic loci in the blood of women with established control values obtained from normal non-pregnant women according to the same method.

Although fetal/placental expressed RNA has been used for prenatal diagnosis and monitoring, see, e.g., U.S. patent application nos. 09/876,005 and 10/759,783, the identification of markers suitable for the particular RNA type of interest has been found to be unpredictable. Since not all RNA types expressed in the placenta can be detected in maternal blood. For example, the inventors failed to detect certain RNA types derived from the fetus/placenta in maternal blood. Some exemplary undetected types include: NADH dehydrogenase (ubiquinone) flavoprotein 3, 10kDa (NDUFV 3); alpha-fetoprotein (AFP); e 1-haemoglobin (HBE1) and phospholipase A2 type IIA (platelets, synovial fluid) (PLA2G 2A).

II preparation of blood samples

A. Obtaining a blood sample

The first step in practicing the invention is to obtain a biological sample, such as a blood sample, from a pregnant woman of gestational age suitable for receiving the test of the invention, or from a woman to be tested for pregnancy. As mentioned above, the appropriate gestational age will vary depending on the condition being detected and the RNA markers sometimes used. Blood collection from women was performed according to standard procedures in hospitals or clinics. An appropriate amount of peripheral blood, e.g., 3-20ml, is collected and stored according to standard procedures prior to subsequent processing.

B. Preparation of plasma or serum samples

Serum and plasma from women's blood are suitable for the present invention and can be obtained by well-known methods. For example, a woman's blood may be placed in a container containing EDTA or a specific commercial product, such as Vacutainer SST (Becton Dickinson, Franklin Lakes, N.J., USA) to prevent blood clotting, and plasma may be obtained from whole blood by centrifugation. In another aspect, serum can be obtained by centrifuging blood after coagulation. Centrifugation is typically carried out at a suitable speed, e.g., 1,500-3,000 Xg, in a refrigerated environment, e.g., 4-10 ℃. The plasma or serum may also be subjected to additional centrifugation steps before being transferred to a new tube for RNA extraction. In certain applications of the invention, plasma or serum may be the preferred sample type. While in other applications of the invention, whole blood may be preferred. And in other applications, other components of the blood are preferred.

III quantitative determination of the amount of RNA in the blood of women

RNA extraction

There are various methods for extracting RNA from biological samples. Common methods for RNA preparation can be used (e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual3d ed., Molecular Cloning: A Laboratory Manual3 rd edition, 2001); various commercially available reagents or Kits, such as Trizol reagent (Invitrogen, Carlsbad, Calif., USA), OligoteX Direct mRNA Kits (Qiagen, Valencia, Calif., USA), RNeasy Mini Kits (Qiagen, Hilden, Germany), andSeries9600^TM(Promega, Madison, Wis., USA) to obtain RNA from women's blood samples. These methods can also be combined to extract RNA.

[0061] In certain applications, it is preferred to remove all or most of the contaminating DNA from the RNA preparation. Therefore, in the amplification and quantification steps, the sample should be handled carefully, the DNase treatment performed thoroughly and appropriate negative controls set.

B. PCR-based quantitative determination of RNA levels

Once RNA has been extracted from a woman's blood sample, the amount of RNA derived from the genetic locus of interest, such as COL6A1, COL6A2, APP, ATP5O or LEP, can be quantified. Preferred methods for determining the level of said RNA are amplification-based methods, such as PCR.

Before performing the amplification step, a DNA copy (cDNA) of the RNA of interest must be synthesized. This can be done by reverse transcription, either as a separate step or as a homogeneous reverse transcription-polymerase chain reaction modified for polymerase chain reaction for amplification of RNA. Suitable methods for PCR amplification of ribonucleic acids can be found in diagnostic molecular biology, Romero and Rotbart: principles and Applications (Diagnostic Molecular Biology: Principles and Applications) 401-; edited by Persing et al, Mayo Foundation, Rochester, MN, 1993; egger et al, j.clin.microbiol.33: 1442-1447, 1995; and U.S. patent No. 5,075,212.

PCR methods are well known in the art and need not be described herein. For a review of PCR methods, reference may be made to the experimental methods and primer design principles, for example, Innis et al, "PCR experimental methods: guidelines for Methods and applications (PCR Protocols), Academic Press, Inc. N.Y., 1990. PCR reagents and procedures are also available from retailers, such as Roche molecular Systems.

In most cases, PCR is performed in an automated process using thermostable enzymes. In this process, the temperature of the reaction mixture is generally self-cycling between the denaturation zone, the primer annealing zone, and the extension reaction zone. In some experimental approaches, the annealing zone may be combined with the extension reaction zone. Machines suitable for this purpose are commercially available.

Although the RNA amplification method typically employed in the practice of the present invention is PCR. However, one skilled in the art will recognize that amplification of these RNA types in maternal blood can also be accomplished by other well-known methods, such as Ligase Chain Reaction (LCR), transcription-mediated amplification, and self-sustained sequence replication or nucleic acid sequence-based amplification (NASBA), each of which can provide sufficient amplification. The amount of RNA marker in maternal blood can also be quantified using recently developed branched DNA techniques. For a review of branched RNA signal amplification for direct quantification of nucleic acid sequences in clinical samples, see Nolte, adv. 201-235, 1998.

C. Other methods of quantification

Other standard techniques known to those skilled in the art can also be used to determine the type of RNA of interest. Although the amplification step typically precedes the assay step, amplification may not be required in the methods of the invention. For example, whether performed before or after the amplification step, the type of RNA of interest can be identified by size separation (e.g., gel electrophoresis). After running through a well-known agarose or polyacrylamide gel electrophoresis and ethidium bromide labeling (see, e.g., Sambrook and Russell, supra), the presence of the same band as a standard control can indicate the presence of target RNA, and the amount of the band can be compared to the standard based on the intensity of the band. Alternatively, based on the intensity of the signal generated by the probe, oligonucleotide probes specific for RNA transcribed from genetic loci such as COL6A1, COL6A2, APP, ATP5O or LEP can be used to detect the presence of these RNA types, and comparison to the standard indicates the amount of the RNA.

Sequence-specific probe hybridization is a well-known method for detecting specific nucleic acids in a context containing other nucleic acid types. Under sufficiently stringent hybridization conditions, the probe specifically hybridizes only to substantially complementary sequences. The stringency of the hybridization conditions can be relaxed to allow for varying amounts of sequence mismatches.

There are a variety of hybridization formats in the art, including liquid phase, solid phase or mixed phase hybridization assays. The following article provides a review of various hybridization assay formats: singer et al, Biotechniques (biotechnology) 4: 230, 1986; haase et al, Methods in Virology, pp.189-226, 1984; wilkinson, In situ Hybridization, Wilkinson ed., IRL Press, Oxford University Press, Oxford; and Hames and higgins eds, Nucleic Acid Hybridization: a Practical Approach (nucleic acid hybridization: manipulation method), IRL Press, 1987.

The hybridization complex can be assayed according to known methods, which are not essential to the present invention. Nucleic acid probes that specifically hybridize to the target nucleic acid, i.e., the RNA type of interest or the amplified DNA, can be labeled using any method commonly used to determine the presence of hybridized nucleic acids. A common assay method is that of³H，¹²⁵I，³⁵S，¹⁴C or³²P, etc. labeled probes are subjected to autoradiography. The choice of radioisotope is based on research preferences, taking into account factors including ease of synthesis, stability and half-life of the isotope. Other labels include labels of compounds (e.g., biotin and digoxigenin) that bind to fluorophores, chemiluminescent agents, and enzyme-labeled anti-ligands (Antilags) or antibodies. Alternatively, the probe may be conjugated directly to a label such as a fluorophore, chemiluminescent agent or enzyme. The choice of label depends on the sensitivity required, the ease of conjugation with the probe, stability requirements and the equipment available.

Probes and primers necessary for the practice of the invention can be synthesized and labeled using well-known techniques. The method may be performed according to methods first described by Beaucage and carothers, Tetrahedron letters, 22: 1859 solid phase phosphoramidite triester method described in 1862, 1981, e.g., needleham-VanDevanter et al, Nucleic Acids Res.12: 6159-6168, 1984, oligonucleotides used as probes and primers were chemically synthesized using an automated synthesizer. Such as Pearson and Regnier, j.chrom, 255: 137-149, 1983, oligonucleotides can be purified by native acrylamide gel electrophoresis or anion exchange High Performance Liquid Chromatography (HPLC).

Establishment of Standard controls

To establish a standard control, a group of healthy pregnant women carrying a healthy fetus is first selected. These women should have similar gestational ages that are within the appropriate stage of pregnancy for the detection of conditions such as preeclampsia and fetal chromosomal aneuploidy (including trisomy 18 and trisomy 21) using the methods of the invention. Similarly, samples from a group of healthy, non-pregnant women can be used to establish a standard control.

The health status of selected pregnant women and the foetuses they carry should be confirmed using established, routinely used methods including monitoring the blood pressure of the woman, recording the onset of labour, foetal genetic analysis using CVS and amniotic fluid diagnosis.

Furthermore, the selection of healthy pregnant women carrying a healthy fetus or healthy non-pregnant women on a suitable group scale allows the average amount of RNA derived from the genetic loci indicated in the present application calculated from said group to be a reasonable representation of the normal or average amount in a normal population of healthy pregnant women carrying a healthy fetus or healthy non-pregnant women. Preferably, the selected group contains at least 10 women.

Once the average of the amount of said fetal/placental derived RNA is established based on the individual values determined for each woman in the selected group, this value is considered as a criterion for the type of said RNA. Thus, blood samples containing similar amounts of the same type of RNA can be used as standard controls. Solutions containing established average concentrations of the same type of RNA species of interest can also be prepared manually and used as standard controls.

The following examples are provided for illustrative purposes only and not for limiting purposes. Those skilled in the art will readily appreciate that variations and modifications to the various noncritical parameters may be made to achieve substantially similar results.

Examples

The following examples are provided for illustrative purposes only and not for limiting purposes. Those skilled in the art will readily appreciate that changes and modifications to the various noncritical parameters may be made to achieve substantially the same or similar results.

Example 1 genetic loci of chromosome 21 or 18 expressed in placental tissue

Method of producing a composite material

Test subject

Placental tissue and blood samples were collected from informed consented pregnant women in the first three months who were admitted to the obstetrics and gynecology department at the hong kong wils king hospital. The study has been approved by the ethical committee of clinical research.

Sample preparation for microarray analysis

Before the end of the treatment, 5 placental tissue samples were obtained from the pregnant women for the first three months using Chorionic Villus Sampling (CVS). Fetal karyotyping in all cases was subsequently confirmed as normal. Placental tissue samples were stored in RNAlater immediately at the time of collection^TM( Osthole, texas, usa) and stored at-80 ℃ until RNA extraction is performed. The tissue collection was performed while collecting 6 ml of pregnant woman peripheral blood and storing in PAXgene^TMBlood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland). Total RNA was extracted from placental tissue using Trizol reagent (Invitrogen, Carlsbad, california, usa) and purified using RNeasy mini-kit (Qiagen, Hilden, germany) according to the manufacturer's experimental guidelines. Use of PAXgene according to manufacturer's guidelines^TMBlood RNA kit (PreAnalytiX, Hombrechtikon, Switzerland) extracts total RNA from peripheral blood, and also includes DNase treatment (RNase-Free DNase Set, Qiagen, Hilden, Germany).

Gene expression analysis by high density oligonucleotide microarray

For each sample, 10. mu.g of extracted RNA was used for labeling and comparison with the manufacturer's instructionsHuman genome U133A was hybridized to U133B array (Affymetrix, Santa Clara, Calif., USA). After hybridization, each array is washed and incubatedStaining was performed in Fluidics Station 400(Affymetrix, Santa Clara, california, usa). With a general Scanner (Affymetrix, Santa)Clara, California, USA) to scan the chip withMicroarray Suite 5.0 (Affymetrix).

Real-time quantitative RT-PCR

RNA transcripts in placental tissue and maternal blood samples were quantitated using one-step real-time quantitative RT-PCR (QRT-PCR). QRT-PCR analysis of the detection of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been described previously (Ng et al 2002). The sequences of primers (Prooligo, Singapore) and fluorescent probes (Applied Biosystems, Foster City, Calif., USA) for other genes tested are shown in Table 1A. For analysis of placental tissue and maternal buffy coat, relative quantification was performed, in which transcript levels studied were normalized to the corresponding gapdh mrna levels.

The QRT-PCR reaction was set up in a 25. mu.l reaction volume according to the manufacturer's guidelines (EZ rTth RNA PCR reagent set, applied biosystems). The QRT-PCR analysis was performed on a combined thermal cycler and fluorescence detector (ABI Prism 7900HT, Applied Biosystems). The concentrations of the PCR primers and fluorescent probe were 300nM and 100nM, respectively, for all transcripts. Prior to QRT-PCR, DNase I digestion (Invitrogen, Carlsbad, california, usa) was used to remove contaminating DNA from the placental tissue RNA extracts according to the manufacturer's recommendations. Amplification was performed using 17ng of extracted placental RNA. Multiple negative water blanks were included in each analysis.

The heat treatment process is as follows: the reaction was initiated at 50 ℃ for 2 minutes to allow the uracil N-glycosylase included to act, followed by reverse transcription at 60 ℃ for 30 minutes. After 5 min of denaturation at 95 ℃, 40 PCR cycles were performed at 92 ℃ for 15 sec of denaturation and 1 min of annealing/extension at 58 ℃.

Quantitative analysis of placental expression transcripts in maternal blood

A maternal whole blood sample of a normal pregnant woman was collected into an EDTA tube. After centrifugation of the blood sample at 1,600g for 10 minutes at 4 ℃, the buffy coat and plasma fractions were carefully transferred to separate polypropylene tubes. The plasma samples were centrifuged at 16,000g for a further 10 minutes at 4 ℃. The supernatant was collected into a new polypropylene tube. RNA was extracted from the collected maternal plasma as described previously (Ng et al, 2002). Similarly, RNA was extracted from 0.3mL of the buffy coat fraction.

QRT-PCR analysis was performed on the transcripts studied using the same conditions as described above. Each QRT-PCR reaction required the use of 5 microliters of extracted plasma RNA or 10ng of RNA of the buffy coat. Absolute quantification is used to determine the transcript concentration in plasma samples. At a concentration of from 1x10⁷Copy to 1x10¹Copies, calibration curves were prepared by serial dilution of single-stranded synthetic DNA oligonucleotides (Proligo, Singapore) purified by high performance liquid chromatography with the full length of the amplicon. The absolute concentration of the transcript in plasma is described in copies per ml of plasma. The sequences of the synthesized DNA oligonucleotides are shown in Table 1B. The results of the buffy coat fraction are expressed in relative quantification based on normalization to GAPDH.

Statistical analysis

Statistical analysis was performed using Sigma Stat 2.03 software (SPSS).

Results

Identification of placental expressed genes by high density oligonucleotide microarray

Gene expression profiles were obtained for 5 first three month CVS samples by performing independent microarray analysis on each independent sample. Of the approximately 22,000 detectable transcripts in the human genome, U133A and U133B chips (Affymetrix), a total of 7226 gene transcripts were expressed in the CVS samples. We have previously reported that circulating DNA in normal human plasma is derived primarily from hematopoietic cells (Lui)et al, 2002). Thus, we believe that most background maternal nucleic acids in maternal blood also have a source from the hematopoietic part. Considering our objective to identify the placental expressed transcripts in said circulating RNA molecules of maternal plasma, we further obtained a gene expression profile of maternal whole blood and usedMicroarray Suite 5.0 software (Affymetrix) compares the expression profiles to corresponding placental tissue expression profiles. Placental expressed transcripts of early pregnancy were identified by selection of transcripts with elevated expression levels in the CVS tissue in a total of five sets of comparisons with corresponding whole blood samples. After this step is completed, transcripts expressed in maternal blood cells higher than or similar to the placental tissue are removed. Thus, the analysis identified 1245 transcriptomes with placental specificity in the first trimester of pregnancy.

Selection of genetic loci having placental tissue expression and on chromosome 21 or 18

Among the group of placental-specific transcripts identified by the above method, we further searched for transcripts of genes located on chromosome 21. 13 genes located on chromosome 21 were identified as shown in Table 2A. This gene selection strategy is based on the following reasons: the presence of an alteration in the dose of the gene produced by the extra chromosome 21 in the genome of a fetus with trisomy 21 may result in abnormal expression of the gene located on chromosome 21. As we have previously demonstrated, the placenta is an important source of circulating fetal RNA in maternal plasma (Ng et al, 2003), and abnormal placental tissue expression of this target gene by trisomy 21 is reflected by abnormal concentrations of the transcript in maternal blood. Thus, the method for non-invasive prenatal detection of fetal trisomy 21 by analysis of circulating RNA derived from the fetus/placenta is based on the detection of abnormal blood concentrations of those selected transcripts of pregnant women carrying fetuses of trisomy 21 compared to those of women carrying normal fetuses.

A similar strategy was used to identify gene markers for trisomy 18 in a non-invasive prenatal analysis. Expression profiles of CVS and maternal whole blood samples were analyzed using the human genome U133B array (Affymetrix). In the case of maternal blood, the same screening criteria as above were used to determine the set of transcripts with preferential expression in CVS. Placental expressed genes located on chromosome 18 were selected from the transcriptome on the basis of relative placental specificity. Among this group, the transcripts with the highest expression level in CVS were selected, as shown in table 2B.

Validation of microarrays by real-time QRT-PCR

The expression of the labeled placental tissue identified by the above microarray-based strategy was verified using one-step real-time QRT-PCR. GAPDH mRNA, selected transcripts listed in tables 2A and 2B, were assayed for the first trimester CVS tissue of 10 normal pregnancies and 3 trisomy 21 pregnancies. The relative mRNA levels of the genes tested were normalized to the corresponding GAPDH levels using the following equation:

ΔCt_X＝Ct_GAPDH-Ct_X

where Ct represents the threshold cycle, i.e., the number of PCR cycles required for the QRT-PCR reaction of the sample to accumulate fluorescence to reach a predetermined threshold intensity. Delta Ct_XIs the normalized mRNA level of the transcript X tested; ct_GAPDHIs the Ct value of GAPDHmRNA; ct_XIs the Ct value of the transcript X. Since the Ct value is inversely proportional to the logarithm of the amount of the template mRNA, the Δ Ct value_XThe higher the value, the higher the mRNA level. It was thus confirmed that the transcript tested was expressed and detectable in CVS tissues collected from both normal and trisomy 21-related fetuses.

ADAMTS1mRNA (fig. 1A) (Mann-Whitney test, P0.036) and APP mRNA (fig. 1B) (Mann-Whitney test, P0.036) were found to be statistically significantly upregulated in placental tissue expression in CVS tissues collected from trisomy 21 pregnancy when compared to samples collected from normal pregnancies. These data confirm our hypothesis that genes located on the trisomy are associated with quantitative abnormalities in placental tissue expression, and thus are very useful markers for prenatal evaluation of trisomy 21.

Detectability of said placental expressed transcript in maternal blood

We investigated the detectability of certain transcripts in buffy coat and plasma samples collected from women in the third trimester of pregnancy. All 12 transcripts studied were detectable in both the buffy coat (fig. 2) and plasma samples (data not shown). To test for pregnancy specificity of the transcript, plasma samples were also taken from 10 pregnant women at 24 hours before and after birth. Figures 3A and 3B illustrate that COL6a1 and COL6a2mRNA were rapidly cleared in postpartum maternal plasma (Wilcoxon test, both P < 0.05), while the corresponding GAPDH mRNA levels remained unchanged (data not shown, Wilcoxon test, P ═ 1.000). The clearance of COL6a1 and COL6a2mRNA in postpartum maternal plasma suggests that the placenta is the most prominent tissue source for these transcripts.

Conclusion

The present invention identifies transcripts expressed in placental tissue in the first trimester of pregnancy using a microarray-based method. Based on the selection of the gene expressed by the placenta located on chromosome 21, 13 transcripts for prenatal testing of chromosome 21 trisomy were identified. Similarly, the RNA marker for prenatal detection of chromosome 18 trisomy was also determined by selection of placental transcripts encoded on chromosome 18.

The present invention verifies the detectability of the transcripts tested in normal and non-euploid placental tissues by the real-time QRT-PCR method. For example, ADAMTS1 has abnormal expression with APP mRNA in placental tissue of trisomy 21 pregnancy. In addition, all of the target gene mRNA is detectable in the maternal buffy coat and plasma. These data demonstrate that our marker selection strategy is capable of identifying RNA types that are aberrantly expressed in trisomy 21 and is detectable in maternal circulation, which may facilitate the development of a non-invasive prenatal diagnostic strategy for fetal trisomy 21. For example, non-invasive prenatal testing of trisomy 21 can be accomplished based on the determination of abnormal concentrations of the RNA marker for trisomy 21 pregnancy compared to corresponding concentrations in normal pregnancy. Alternatively, non-invasive prenatal diagnosis may be performed based on a relative quantitative comparison of the different molecular forms of one or more of the transcripts in maternal plasma. Trisomy 18 can also be diagnosed by measurement of RPL17mRNA in maternal plasma.

Example 2 comparison of genes with elevated expression in the placenta of trisomy 21 pregnancy with genes of normal pregnancy

Method of producing a composite material

Test subject

All placental tissue and blood samples were collected from informed consented pregnant women in the first trimester of pregnancy who had a gynecological visit to the hong kong wils kindsman hospital. The study has been approved by the ethical committee of clinical research.

In the first part of the study, gene expression profiles of normal and trisomy 21 pregnant placental tissues were identified using oligonucleotide microarrays. Placental tissue samples were obtained from pregnant women for the first three months using Chorionic Villus Sampling (CVS). 5 women with normal pregnancy (10-12 weeks of gestational age) and 3 women with trisomy 21 (12-13 weeks of gestational age) were recruited and then individually subjected to fetal karyotype analysis and confirmed diagnosis. In the second part of the study, QRT-PCR was used to validate gene expression profiles generated by the oligonucleotide microarray experiments. For the study, CVS was used from 3 women with trisomy 21 fetuses (13-14 weeks gestational age) and 5 normal pregnant women (9-13 weeks gestational age).

Sample preparation for microarray analysis

CVS samples were stored in RNAlater immediately after collection^TM( Osthole, texas, usa) and stored at-80 ℃ until RNA extraction is performed. For five pregnant women with normal pregnancy, 6 ml of peripheral blood was collected and stored in PAXgene while tissue collection was performed^TMBlood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland). Total RNA was extracted from placental tissue using Trizol reagent (Invitrogen, Carlsbad, california, usa) and purified using RNeasy mini-kit (Qiagen, Hilden, germany) according to the manufacturer's experimental guidelines. Use of PAXgene according to manufacturer's guidelines^TMBlood RNA kit (PreAnalytiX, Hombrechtikon, Switzerland) extracts total RNA from peripheral blood, and also includes DNase treatment (RNase-Free DNase Set, Qiagen, Hilden, Germany).

Gene expression analysis by high density oligonucleotide microarray

The extracted 10. mu.g of RNA were labeled and combined with each sample according to the manufacturer's instructionsHuman genome U133A was hybridized to U133B array (Affymetrix, Santa Clara, Calif., USA). After hybridization, each array is washed and incubatedStaining was performed in fluidics station 400(Affymetrix, Santa Clara, california, usa). The chips were scanned using a general Scanner (Affymetrix, Santa Clara, Calif., USA) using a ScannerMicroarray Suite 5.0(Affymetrix) for analysis.

Real-time quantitative RT-PCR

RNA transcripts in placental tissue and maternal blood samples were quantitated using one-step real-time quantitative RT-PCR (QRT-PCR). QRT-PCR analysis of the detection of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been described previously (Ng et al 2002). The sequences of other gene-detecting primers (Prooligo, Singapore) and TaqMan Minor Groove Binding (MGB) fluorescent probes (Applied Biosystems, Foster City, Calif., USA) are shown in Table 3. Relative quantification was used to describe the amount of RNA expressed, where the transcript levels studied were normalized to the corresponding GAPDH mRNA levels.

The QRT-PCR reaction was set up in a 25. mu.l reaction volume according to the manufacturer's guidelines (EZ rTth RNA PCR reagent set, applied biosystems). The QRT-PCR analysis was performed on a combined thermal cycler and fluorescence detector (ABI Prism 7900HT, Applied Biosystems). The concentrations of the PCR primers and fluorescent probe were 300nM and 100nM, respectively, for all transcripts. Prior to QRT-PCR, the fetal disc tissue RNA extracts were freed of contaminating DNA using DNase I digestion (Invitrogen, Carlsbad, california, usa) according to the manufacturer's recommendations. Amplification was performed using 17ng placental RNA extract. Multiple negative water blanks were included in each analysis.

The heat treatment process is as follows: the reaction was initiated at 50 ℃ for 2 minutes to allow the uracil N-glycosylase included to act, followed by reverse transcription at 60 ℃ for 30 minutes. After 5 min of denaturation at 95 ℃, 40 PCR cycles were performed at 92 ℃ for 15 sec of denaturation and 1 min of annealing/extension at 58 ℃.

Quantitative analysis of chromosome 21 trisomy associated with placental transcripts in maternal blood

Maternal whole blood samples from normal pregnant women were collected in EDTA tubes. After centrifugation of the blood sample at 1,600g for 10 minutes at 4 ℃, the plasma fraction was carefully transferred to an empty polypropylene tube. The plasma samples were centrifuged at 16,000g for a further 10 minutes at 4 ℃. The supernatant was collected into a new polypropylene tube. RNA was extracted from the collected maternal plasma as described previously (Ng et al, 2002). QRT-PCR analysis was performed on the study transcripts using the same conditions as described above. Each QRT-PCR reaction required the use of 5 microliters of extracted plasma RNA.

Statistical analysis

Statistical analysis was performed using Sigma Stat 2.03 software (SPSS).

Results

Microarray-based identification of abnormal placental tissue expression genes in aneuploid pregnancy

Gene expression profiles were obtained for 5 first three month CVS samples by performing independent microarray analysis on each individual sample. We have previously reported that circulating DNA in normal human plasma is derived primarily from hematopoietic cells (Lui et al, 2002). Therefore, we hypothesize that in maternal blood the majority of background maternal nucleic acids also originate from the hematopoietic part. Considering that the ultimate goal of this study was to identify placental expressed transcripts that were fetal-specific in circulating RNA molecules in maternal plasma, we further obtained gene expression profiles of paired maternal whole blood and compared these expression profiles to those in corresponding 5 normal pregnancy samples. Use is thatMicroarray Suite 5.0 software (Affymetrix). By selecting for transcripts with elevated expression levels in the CVS tissue, transcripts with relative placental specificity were identified when compared to corresponding whole blood samples in all 5 group comparisons. After this step is completed, transcripts expressed in maternal blood cells higher than or similar to the CVS tissue are removed. Thus, the analysis identified a transcriptome with placental specificity in the first trimester of pregnancy.

In the next step, transcripts that were aberrantly expressed in placental tissue of aneuploidy pregnancies were identified. Use ofMicroarray Suite 5.0 software (Affymetrix) compares the expression profile of 3 CVS tissues from chromosome 21 trisomy with the placenta-specific genome in 5 gestational age-matched normal pregnancies described above. Each gene expression signal of the 3 aneuploidy CVS samples was individually compared to each of 5 normal CVS samples, using the normal placental tissue expression profile as baseline. A total of 15 comparisons were made, counting (I-counts) the number of comparisons that were up-regulated in expression in the aneuploid placenta for each gene interrogated. The fold change in expression level was calculated and converted to log₂Value (signal Log ratio, SLR). Selecting transcripts if (i) said transcripts in an aneuploid placenta are up-regulated if said Log ratio of signal is at least 0.4 when compared to a normal placenta; and (ii) the upregulation is consistent with upregulation given by more than half of the comparisons (I-count ≧ 8). Table 4 summarizes the microarray results for the 3 transcripts, namely EGF-containing fibulin-like extracellular matrix protein1 (EGF-stabilizing fibulin-like extracellular matrix protein1) (EFEMP1), transferrin receptor p90 CD71(TFRC) and ATP5O, which are the most highly regulated genes preferentially expressed in the placenta in the gene panel of trisomy 21 pregnancy. Validation of microarrays by real-time QRT-PCR

The transcripts expressed by the abnormal placenta tissues of chromosome 21 trisomy 3 pregnancies identified by the microarray experiment are verified by adopting one-step real-time QRT-PCR. The mRNA levels of 3 transcripts and GAPDH were quantified in CVS tissues collected from 3 trisomy 21 pregnancies and 5 normal pregnancies of matching gestational age. The relative mRNA levels of the genes tested were normalized to the corresponding GAPDH levels using the following equation:

ΔCt_X＝Ct_GAPDH-Ct_X

where Ct represents the threshold cycle, i.e., the number of PCR cycles required for the QRT-PCR reaction of the sample to accumulate fluorescence to reach a predetermined threshold intensity. Delta Ct_XIs the normalized mRNA level of the transcript X tested; ct_GAPDHIs the Ct value of GAPDHmRNA; ct_XIs the Ct value of the transcript X. Since the Ct value is inversely proportional to the logarithm of the amount of the template mRNA, the Δ Ct value_XThe higher the value, the higher the mRNA level.

QRT-PCR revealed that EFEMP1mRNA (FIG. 4A), TFRC mRNA (FIG. 4B) and ATP5O mRNA (FIG. 4C) were indeed up-regulated in CVS tissues of trisomy 21 pregnancy when compared to CVS samples taken from normal pregnancies. Abnormal placental tissue expression of the three transcripts in aneuploidy pregnancies demonstrates their role as RNA markers in prenatal testing of chromosome 21 trisomy.

Detectability of the RNA marker in maternal blood

Plasma samples from normal pregnant women were assayed for ATP5O mRNA. At 24 hours post-partum, there was a statistically significant decrease in the concentration of ATP5OmRNA (FIG. 4D; Wilcoxon, P < 0.05), which was detectable in maternal plasma (FIG. 4D). These data indicate that placenta is an important tissue source for ATP5OmRNA in maternal plasma.

Conclusion

The present invention identifies transcripts with aberrant expression in placental tissue from chromosome 21 trisomy using microarray-based methods. The three transcripts, EFEMP1, TFRC and ATP5O, were identified by microarray technology and further validated for their abnormal properties of expression in placental tissue of trisomy 21 gestation by QRT-PCR. These data thus indicate that these 3 transcripts can be used as RNA markers for prenatal detection of chromosome 21 trisomy. The detectability of ATP5OmRNA in maternal plasma indicates the applicability of this transcript to non-invasive prenatal detection of foetal trisomy 21. For example, non-invasive prenatal testing of trisomy 21 can be accomplished based on abnormal concentrations as compared to corresponding concentrations in normal pregnancy for the RNA markers determined in trisomy 21 pregnancy.

Example 3 comparison of genes aberrantly expressed in the placenta of pregnancies affected by preeclampsia with the corresponding genes of normal pregnancies

Method of producing a composite material

Test subject

All placental tissue and blood samples were collected from informed consented pregnant women in the third trimester of pregnancy who had a gynecological visit to the hong kong wils kindsman hospital. The study has been approved by the ethical committee of clinical research.

In the first part of the study, gene expression profiles of placental tissues from normal and Preeclamptic (PET) pregnancies were identified using oligonucleotide microarrays. Immediately after cesarean section, placental tissues were collected from 5 PET pregnant women (37-40 weeks gestational age) and 5 normal pregnant women (38-40 weeks gestational age). Peripheral blood was collected prior to delivery. In the second part of the study, QRT-PCR was used to validate gene expression profiles generated by the oligonucleotide microarray experiments. Immediately after cesarean section, placental tissues were collected from 10 PET pregnant women (25-40 weeks gestational age) and 10 normal pregnant women (37-39 weeks gestational age). For a woman without a history of hypertension, the woman is considered to have preeclampsia if diastolic blood pressure continues to increase, once > 110mm Hg, or two or more times > 90mm with at least 4 hour intervals, and a significant proteinuria condition exists. Significant proteinuria was defined as > 0.3 g/day proteinuria or > 2+ in dipstick tests of two cleanly collected mid-stream urine samples taken at least 4 hour intervals.

Sample preparation for microarray analysis

Placental tissue samples were stored in RNAlater immediately after collection^TM( Osthole, texas, usa) and stored at-80 ℃ until RNA extraction is performed. 6 ml of peripheral blood were collected simultaneously with tissue collection and stored in PAXgene^TMBlood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland). Total RNA was extracted from placental tissue using Trizol reagent (Invitrogen, Carlsbad, california, usa) and purified using RNeasy mini-kit (Qiagen, Hilden, germany) according to the manufacturer's experimental guidelines. Use of PAXgene according to manufacturer's guidelines^TMBlood RNA kit (PreAnalytiX, Hombrechtikon, Switzerland) extracts total RNA from peripheral blood, and also includes DNase treatment (RNase-Free DNase Set, Qiagen, Hilden, Germany).

Gene expression analysis by high density oligonucleotide microarray

The extracted 10. mu.g of RNA were labeled and combined with each sample according to the manufacturer's instructionsHuman genome U133A was hybridized to U133B array (Affymetrix, Santa Clara, Calif., USA). After hybridization, each array is washed and incubatedStaining was performed in Fluidics Station 400(Affymetrix, Santa Clara, california, usa). The chips were scanned using a general Scanner (Affymetrix, Santa Clara, Calif., USA) using a ScannerMicroarray Suite 5.0 (Affymetrix).

Real-time quantitative RT-PCR

RNA transcripts in placental tissue and maternal plasma samples were quantitated using one-step real-time quantitative RT-PCR (QRT-PCR). QRT-PCR analysis of the detection of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been described previously (Ng et al 2002). The sequences of other gene-detecting primers (Prooligo, Singapore) and TaqMan Minor Groove Binding (MGB) fluorescent probes (Applied Biosystems, Foster City, Calif., USA) are shown in Table 5. Relative quantification was used to describe the amount of RNA expressed, where the transcript levels studied were normalized to the corresponding GAPDH mRNA levels.

The QRT-PCR reaction was set up in a 25. mu.l reaction volume according to the manufacturer's guidelines (EZ rTth RNA PCR reagent set, applied biosystems). The QRT-PCR analysis was performed on a combined thermal cycler and fluorescence detector (ABI Prism 7900HT, Applied Biosystems). The concentrations of the PCR primers and fluorescent probe were 300nM and 100nM, respectively, for all transcripts. Prior to QRT-PCR, DNase I digestion (Invitrogen, Carlsbad, california, usa) was used to remove contaminating DNA from the placental tissue RNA extracts according to the manufacturer's recommendations. Amplification was performed using 17ng placental RNA extract. Multiple negative water blanks were included in each analysis.

The heat treatment process is as follows: the reaction was initiated at 50 ℃ for 2 minutes to allow the uracil N-glycosylase included to act, followed by reverse transcription at 60 ℃ for 30 minutes. After 5 min of denaturation at 95 ℃, 40 PCR cycles were performed at 92 ℃ for 15 sec of denaturation and 1 min of annealing/extension at 56 ℃.

Quantitative analysis of placental transcripts associated with preeclampsia in maternal blood

A maternal whole blood sample of a pregnant woman was collected in an EDTA tube. After centrifugation of the blood samples at 1,600g for 10 minutes at 4 ℃, the plasma fractions were carefully transferred to empty polypropylene tubes. The plasma samples were centrifuged at 16,000g for a further 10 minutes at 4 ℃. The supernatant was collected into a new polypropylene tube. RNA was extracted from the collected maternal plasma as described previously (Ng et al, 2002). QRT-PCR analysis was performed on the transcripts studied using the same conditions as described above. Each QRT-PCR reaction required the use of 5 microliters of extracted plasma RNA.

Statistical analysis

Statistical analysis was performed using Sigma Stat 2.03 software (SPSS).

Results

Identification of genes with aberrant placental tissue expression in pregnancies by microarray-based methods

Our final aim was to develop a method for diagnosing PET by the determination of PET-related transcripts in maternal plasma. The selection of genes therefore begins with the identification of transcripts preferentially expressed in the PET placenta rather than in maternal peripheral blood cells. The design of this strategy is based on our previous findings that in normal individuals, the placenta is an important source of circulating fetal RNA in maternal blood and the hematopoietic system is a major source of plasma DNA (Lui et al, 2002). Gene expression profiles were determined for 5 PET placental tissues and their corresponding peripheral blood samples by oligonucleotide microarray. To identify placental expressed genes, transcripts were selected that had expression in at least 4 of the 5 placental tissue samples analyzed. Genes having expression in maternal blood cells are then removed by positive selection of transcripts whose expression levels are either "absent" from all 5 peripheral blood samples or "elevated" in the placenta when compared to the whole blood samples corresponding to all 5 paired placenta and maternal blood samples. Thus, transcripts having similar or higher levels of expression in maternal blood cells as in placental tissue are removed. Selected sets of transcripts are obtained by these procedures, which are preferably expressed in placental tissue.

In the next step, transcripts with aberrant expression in PET placenta were identified. Use ofMicroarray Suite 5.0 software (Affymetrix) expression profiling of each placenta in 5 PET aliquots with pregnancyThe placenta expression profiles of normal pregnancies matched for age were compared. Expression signals for the placenta-specific genes identified from the 5 above-described PET pregnancy samples were individually compared to each of the 5 normal placenta samples, using the normal placenta tissue expression profile as a baseline. A total of 25 comparisons were made, counting (I-counts) the number of comparisons showing up-regulation of expression in the PET placenta for each gene interrogated. Fold change in expression level was calculated and converted to log₂Value (signal Log ratio, SLR). The transcripts are then selected if (i) said transcripts in the PET placenta are upregulated to the extent that said Log-of-signal ratio is at least 0.4 (1.3 fold change in expression) when compared to normal placenta; and (ii) the upregulation is consistent with upregulation given by more than half of the comparisons (I-count ≧ 13). Table 6 summarizes the results for 10 identified transcripts with up-regulated preferential expression in PET-pregnant placenta.

Validation of microarrays by real-time QRT-PCR

The PET-related transcripts selected from the microarray experiments were validated using one-step real-time QRT-PCR. GAPDH mRNA concentrations were determined in each placental tissue collected from 10 PET and normal pregnancies of matching gestational age. This GAPDH mRNA concentration was used to normalize the transcript levels between different samples. The expression levels of the 10 transcripts in the two sets of pregnant placental tissue samples identified by microarray analysis were then compared. The relative mRNA levels of the genes tested were normalized to the corresponding GAPDH levels using the following equation:

ΔCt_X＝Ct_GAPDH-Ct_X

where Ct represents the threshold cycle, i.e., the number of PCR cycles required for the QRT-PCR reaction of the sample to accumulate fluorescence to reach a predetermined threshold intensity. Delta Ct_XIs the normalized mRNA level of the transcript X tested; ct_GAPDHIs the Ct value of GAPDHmRNA; ct_XIs the Ct value of the transcript X. Due to Ct value andthe logarithm of the amount of the template mRNA is inversely proportional, so that the Δ Ct_XThe higher the value, the higher the mRNA level.

When normal pregnancies were compared by QRT-PCR analysis, significant upregulation of Leptin (LEP) and sialic acid binding Ig-like lectin 6 (SIGLEC6) mRNA was demonstrated in PET placenta (Leptin and SIGLEC6mRNA, respectively, in FIGS. 5A and 5B) (Mann-Whitney test, P < 0.05 for both cases). These data confirm that our transcript selection strategy is able to identify markers that are aberrantly expressed in PET placental tissue.

Detectability of the PET-associated RNA marker in maternal blood

Measurements of LEP and INHBA mRNA were performed by QRT-PCR on 25 healthy and 26 plasma samples affected by PET during the third trimester of pregnancy. The LEP and INHBA concentrations in maternal plasma in pregnancies affected by PET were significantly elevated when compared to normal pregnancies (LEP: figure 6A, Mann-Whitney, P ═ 0.017; INHBA: figure 6B, Mann Whitney, P ═ 0.006).

Conclusion

The present invention uses microarray-based methods to identify transcripts with differential expression in PET placenta and considers them as important markers for diagnosing PET. Among the list of PET-related transcripts identified by microarray analysis, 10 were selected for the most aberrantly expressed transcripts in the PET placenta compared to normal pregnancy. Real-time QRT-PCR also demonstrated that expression of both LEP and SIGLEC6 was significantly upregulated in PET placenta when compared to normal placenta. The INHBA and LEP levels in maternal plasma in PET pregnancies were also significantly increased compared to normal pregnancies, thus illustrating the possibility of using these markers for non-invasive prenatal assessment of PET.

Example placenta-specific PLAC4mRNA in trisomy 421 and in plasma from normal pregnant women

Detection of PLAC4mRNA and determination of pregnancy specificity

PLAC4mRNA was determined in maternal plasma using real-time QRT-PCR analysis. In addition, PLAC4mRNA is cleared from the maternal plasma after birth. Thus, PLAC4mRNA in maternal plasma is fetal in origin and specific for pregnancy.

Sample collection and processing

Peripheral blood samples were collected from 5 non-pregnant women, 5 first trimester and 8 third trimester. Peripheral blood was also collected from 6 women in the third trimester of pregnancy 24 hours before and after delivery. The blood sample was collected in EDTA tubes. Plasma samples were collected as described in example 1. RNA was extracted from maternal plasma according to the procedure described in example 1.

Development of real-time QRT-PCR analysis

QRT-PCR analysis of PLAC4mRNA was developed as described in example 1. The sequences of the primers (Integrated DNA Technologies, Coralville, IA, USA) and TaqMan Minor Groove Binding (MGB) fluorescent probes (Applied Biosystems, Foster City, Calif., USA) and calibrators (Prooligo, Singapore) are shown in Table 7.

The QRT-PCR reaction was set up in a 25. mu.l reaction volume according to the manufacturer's guidelines (EZ rTth RNA PCR reagent set, applied biosystems). In ABI7900HT (applied biosystems) the QRT-PCR analysis was performed. The concentrations of the PCR primers and the fluorescent probe were 400nM and 100nM, respectively. Mu.l of the extracted RNA was used for amplification. The heat treatment process comprises the following steps: the reaction was initiated at 50 ℃ for 2 minutes and then reverse transcribed at 60 ℃ for 30 minutes. After 5 minutes of denaturation at 95 ℃, denaturation at 95 ℃ for 15 seconds and at 60 ℃ for 1 minute, 45 PCR cycles were performed.

PLAC4mRNA can be detected in maternal plasma and is pregnancy specific

PLAC4mRNA was detected in all first and last trimester pregnant women, but not in non-pregnant individuals (fig. 7). The median concentration of PLAC4mRNA in plasma of the first and last trimesters of pregnancy was 299.6 copies/ml and 529.3 copies/ml, respectively. The pregnancy specificity of the circulating PLAC4mRNA was determined. The median concentration of PLAC4mRNA in prenatal plasma samples was 500.0 copies/ml. The transcript was not detectable in all postpartum plasma samples (fig. 8).

Comparison of circulating PLAC4mRNA in euploid and trisomy 21 pregnancies

Circulating PLAC4mRNA concentrations were compared between karyotyping normal and gestation in trisomy 21. Plasma samples were collected from 29 pregnant women with euploid fetuses and 5 pregnant women with trisomy 21 fetuses in the first and middle trimesters of pregnancy. PLAC4mRNA concentration in the plasma samples was then determined by real-time one-step RT-PCR. PLAC4mRNA concentrations were determined for all trisomy plasma samples. The median values in trisomy 21 and normal pregnancy were 5581 copies/ml and 4836 copies/ml, respectively. Because of the small number of samples, no statistically significant differences were established between normal and trisomy 21 pregnancies.

Example 5 comparison of genes aberrantly expressed in the placenta of pregnancies affected by preeclampsia with the corresponding genes in normal pregnancies

Method of producing a composite material

Test subject

All placental tissue and blood samples were collected from informed consented pregnant women in the third trimester of pregnancy who had a gynecological visit to the hong kong wils kindsman hospital. The study has been approved by the ethical committee of clinical research.

In the first part of the study, gene expression profiles of placental tissues from normal and Preeclamptic (PET) pregnancies were identified using oligonucleotide microarrays. Immediately after cesarean section, placental tissues were collected from 5 PET pregnant women (37-40 weeks gestational age) and 5 normal pregnant women (38-40 weeks gestational age). Peripheral blood was collected prior to delivery. In the second part of the study, QRT-PCR was used to validate gene expression profiles generated by the oligonucleotide microarray experiments. Immediately after cesarean section, placental tissues were collected from 6 PET-pregnant women (gestational age 30-39 weeks) and 6 normal-pregnant women (gestational age 37-39 weeks). For a woman without a history of hypertension, the woman is considered to have preeclampsia if diastolic blood pressure continues to increase, once > 110mm Hg, or two or more times > 90mm with at least 4 hour intervals, and a significant proteinuria condition exists. Significant proteinuria was defined as > 0.3 g/day proteinuria or > 2+ in dipstick tests of two cleanly collected mid-stream urine samples taken at least 4 hour intervals.

Sample preparation for microarray analysis

Placental tissue samples were stored in RNAlater immediately after collection^TM( Osthole, texas, usa) and stored at-80 ℃ until RNA extraction is performed. 6 ml of peripheral blood were collected simultaneously with tissue collection and stored in PAXgene^TMBlood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland). Total RNA was extracted from placental tissue using Trizol reagent (Invitrogen, Carlsbad, california, usa) and purified using RNeasy mini-kit (Qiagen, Hilden, germany) according to the manufacturer's experimental guidelines. Use of PAXgene according to manufacturer's guidelines^TMBlood RNA kit (PreAnalytiX, Hombrechtikon, Switzerland) extracts total RNA from peripheral blood, and also includes DNase treatment (RNase-Free DNase Set, Qiagen, Hilden, Germany).

Gene expression analysis by high density oligonucleotide microarray

The extracted 10. mu.g of RNA were labeled and analyzed for each sample according to the manufacturer's instructionsAndhuman genome U133A was hybridized to U133B array (Affymetrix, Santa Clara, Calif., USA). After hybridization, each array is washed and incubatedStaining was performed in fluidics station 400(Affymetrix, Santa Clara, california, usa). The chips were scanned using a GeneArray Scanner (Affymetrix, Santa Clara, Calif., USA) and analyzed using GeneSpring v 7.2(Agilent Technologies, Palo Alto, Calif., USA).

Mining/analysis of microarray gene expression data

The microarray data were imported in the CEL format into GeneSpring v 7.2(Agilent technologies). Data mining was performed independently on normal and PET pregnant samples (placental tissue and maternal blood cells). In each set of pregnancies, genes were first determined which were expressed relatively higher in placental tissue samples than in maternal blood cells. The microarray raw data of 5 placenta and paired maternal blood cells were normalized sequentially using the following steps: (1) processing the raw data with a Robust Multi-chip Average with GC-content background correction (GC-RMA); (2) performing data conversion whereby microarray values below 0.001 are all set to 0.001; and (3) dividing the signal intensity of each gene by the median value determined in all samples. Genes with statistically significant (P < 0.05) expression in placental tissue or maternal blood cells were then determined. These genes were stored in order of fold difference in expression in placental tissues and in maternal blood cells, according to the purpose of determining transcripts with higher fold difference. Such data mining processes can identify genes that are relatively highly expressed in placental tissue as compared to maternal blood cells.

On the other hand, data mining can also be used to identify genes with higher absolute expression levels in placental tissue. Raw microarray data from the placental tissues were normalized by GC-RMA treatment followed by data transformation with microarray values below 0.001 set to 0.001. Genes were ranked according to normalized expression levels. Placental tissue collected from normal and preeclamptic pregnancies is also independently data mined.

These selected genes were studied extensively if the fold difference of the genes in the PET placenta versus paired maternal blood was higher than that of normal pregnancy, or if those genes had a much higher absolute expression level in PET than in normal placenta, while the expression in placental tissue and maternal blood was at least 200 fold different.

Real-time quantitative RT-PCR

Quantitative determination of RNA transcripts in placental tissues was performed using one-step real-time quantitative RT-PCR. At a concentration of from 2.5x10⁶Copy to 2.5 copies and prepare calibration curves by serial dilution of single-stranded synthetic DNA oligonucleotides purified by high performance liquid chromatography. The sequences of the primers (Prooligo), fluorescent probes (Applied Biosystems) and oligonucleotide calibrators for the genes under investigation are shown in Table 8.

The QRT-PCR reaction was set up in a 50. mu.l reaction volume according to the manufacturer's guidelines (EZ rTth RNA PCR reagent set, applied biosystems). The QRT-PCR analysis was performed on a combined thermal cycler and fluorescence detector (ABI Prism 7900HT, Applied Biosystems). The concentration of the fluorescent probe was 100nM for all transcripts studied. 300nM forward and reverse primers were used for each reaction analyzed for pregnancy-associated plasma protein A (pregnancy-associated plasma protein A), pappalysin1(PAPPA), INHBA and FN1, respectively. 400nM forward and reverse primers were used for each reaction analyzed for LEP, ADAM metallopeptidase domain 12(meltrin alpha) (ADAM12) and pappalysin 2(PAPPA2), respectively. Prior to QRT-PCR, DNase I digestion (Invitrogen) was used to remove contaminating DNA from the placental tissue RNA extracts according to the manufacturer's recommendations. Amplification was performed using 1ng of extracted placental RNA. Multiple negative water blanks were included in each analysis. The RNA concentration of the placenta tissue is expressed in terms of copy number/ng of total RNA in the placenta.

The heat treatment process is as follows: the reaction was initiated at 50 ℃ for 2 minutes to allow the uracil N-glycosylase included to act, followed by reverse transcription at 60 ℃ for 30 minutes. After 5 min denaturation at 95 ℃, denaturation at 92 ℃ for 15 sec and 40 PCR cycles of annealing/extension at 56 ℃ for LEP, ADAM12, PAPPA and INHBA and 57 ℃ for PAPPA2 and FN1 for 1 min were performed.

Statistical analysis

Statistical analysis was performed using Sigma Stat 3.0 software (SPSS).

Results

Genes identified from the microarray analysis include LEP, ADAM12(GenBank accession No.: NM-003474, NM-021641), PAPPA (GenBank accession No.: NM-002581), PAPPA2(GenBank accession No.: NM-020318, NM-021936), INHBA, and FN 1. Placental tissue expression levels of selected transcripts in PET and normal pregnancies were determined using one-step real-time QRT-PCR. The results are shown in FIG. 9. The concentration of LEP, ADAM12, PAPPA2, INHBA and FN1 was higher in the placenta collected from PET pregnancy than in normal pregnancy, while the concentration of PAPPA mRNA was lower in the placenta collected from PET pregnancy.

Conclusion

The present invention uses microarray-based methods to identify transcripts with aberrant expression in PET placenta and considers them as important markers for diagnosing PET. 6 transcripts were selected from the microarray analysis and their characterization of aberrant expression profiles in PET placenta was verified by real-time QRT-PCR.

Reference to the literature

Lui, YYN, Chik, KW, Chiu, RWK, Ho, CY, Lam, CW and Lo, YMD (2002) & major hematopoietic sources of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation (Predominant hematopoietic origin of cell-free DNA in plasma and serum) & Clin Chem48, 421-427.Ng, EKO, Tsui, NBY, Lam, NY, Chiu, RWK, Yu, SC, Wong, SC, Lo, ES, Ranner, TH, Johnson, PJ and Lo, YMD (2002) & existence of filterable and non-filterable mRNA in cancer patients and healthy individuals & gt expression of filtration and mRNA & 48 & gt, clinical research & gt 1212.

Ng, EKO, Tsui, NBY, Lau, TK, Leung, TN, Chiu, RWK, Panesar, NS, Lit, LCW, Chan, KW and Lo, YMD (2003) easy detection of placenta-derived mRNA in maternal plasma (mRNA of placenta origin is sensitive detectable in matrix plasma U.S. A.100, Proc Natl Acad Sci U.S. S.A.4748-.

All patents, patent applications, and other publications cited in this application, including published amino acid or polynucleotide sequences, are hereby incorporated by reference in their entirety for all purposes.

TABLE 1A sequences of primers and probes for real-time QRT-PCR detection of transcripts on chromosome 21 and with placental expression

MGBNFQ: minor groove binding non-fluorescent quenchers; FAM: a fluorescent reporter; TAMRA: fluorescence quenching agent

TABLE 1B oligonucleotide calibrator sequences for absolute quantification of transcripts on chromosome 21 and with placental expression

TABLE 2A microarray detection of placental expression genes located on chromosome 21

Table 2B among the placental expression genes located on chromosome 18, the transcript having the highest expression level in the placenta in the first three months. The genes were determined from the human genome U133B array (Affymetrix).

Table 3 sequences of primers and probes for real-time QRT-PCR determination of aberrantly expressed placental expressed transcripts in chromosome 21 trisomy.

MGBNFQ: minor groove binding non-fluorescent quenchers

Table 4 microarray assays of placental expressed genes with expression differences in trisomy 21 and normal CVS tissues. The genes were detected by human chromosome U133A array (Affymetrix).

TABLE 5 sequences of primers and probes for real-time QRT-PCR detection of preeclampsia-related placental expression transcripts

MGBNFQ: minor groove binding non-fluorescent quenchers; FAM: a fluorescent reporter; TAMRA: fluorescence quenching agent

Table 6 microarray assays of placental expressed genes with expression differences in preeclampsia and normal placental tissues. The genes were detected by the human genome U133A and U133B arrays (Affymetrix).

TABLE 7 sequences of primers, calibrators, and probes for real-time QRT-PCR detection of PLAC4mRNA

MGBNFQ: minor groove binding non-fluorescent quenchers

TABLE 8 sequences of primers, probes and calibrator for real-time QRT-PCR detection of preeclampsia-related placental expression transcripts

MGBNFQ: minor groove binding non-fluorescent quenchers; FAM: a fluorescent reporter; TAMRA: fluorescence quenching agent

List of sequence numbers

Sequence listing

<110> hong Kong Chinese university

<120> markers for prenatal diagnosis and monitoring

<130>10C11284CN

<150>US 60/663,293

<151>2005-03-18

<160>113

<170>PatentIn version 3.3

<210>1

<211>23

<212>DNA

<213> COL6A1 Forward primer

<400>1

gacaaagtca agtccttcac caa 23

<210>2

<211>18

<212>DNA

<213> COL6A1 reverse primer

<400>2

gcgttccaca ccaggttt 18

<210>3

<211>16

<212>DNA

<213> COL6A1 Probe

<400>3

cgcttcatcg acaacc 16

<210>4

<211>21

<212>DNA

<213> COL6A2 Forward primer

<400>4

gatcaaccag gacaccatca a 21

<210>5

<211>19

<212>DNA

<213> COL6A2 reverse primer

<400>5

ccgtaggctt cgtgtttca 19

<210>6

<211>15

<212>DNA

<213> COL6A2 Probe

<400>6

cgcatcatca aggtc 15

<210>7

<211>19

<212>DNA

<213> SOD1 Forward primer

<400>7

cagggcatca tcaatttcg 19

<210>8

<211>18

<212>DNA

<213> SOD1 reverse primer

<400>8

tgcttcccca caccttca 18

<210>9

<211>21

<212>DNA

<213> SOD1 probe

<400>9

cagaaggaaa gtaatggacc a 21

<210>10

<211>21

<212>DNA

<213> ATP50 Forward primer

<400>10

ccctcactac caacctgatc a 21

<210>11

<211>21

<212>DNA

<213> ATP50 reverse primer

<400>11

ccttgggtat tgcttaatcg a 21

<210>12

<211>15

<212>DNA

<213> ATP50 Probe

<400>12

tgcttgctga aaatg 15

<210>13

<211>21

<212>DNA

<213> BTG3 Forward primer

<400>13

gatgtcctga aagcctgtga a 21

<210>14

<211>18

<212>DNA

<213> BTG3 reverse primer

<400>14

ggcaagccca ggtcacta 18

<210>15

<211>15

<212>DNA

<213> BTG3 Probe

<400>15

acagctgcat cttgt 15

<210>16

<211>20

<212>DNA

<213> APP Forward primer

<400>16

aaggaaggca tcctgcagta 20

<210>17

<211>21

<212>DNA

<213> APP reverse primer

<400>17

acattggtga tctgcagttc a 21

<210>18

<211>16

<212>DNA

<213> APP Probe

<400>18

tgccaagaag tctacc 16

<210>19

<211>20

<212>DNA

<213> ATP5J Forward primer

<400>19

cctgtccgaa tcagcatgat 20

<210>20

<211>21

<212>DNA

<213> ATP5J reverse primer

<400>20

tgaccgaatg acagaggaga a 21

<210>21

<211>16

<212>DNA

<213> ATP5J Probe

<400>21

cttcagaggc tcttca 16

<210>22

<211>21

<212>DNA

<213> ADAMTS1 Forward primer

<400>22

ccacaggaac tggaagcata a 21

<210>23

<211>21

<212>DNA

<213> ADAMTS1 reverse primer

<400>23

caagcatggt ttccacatag c 21

<210>24

<211>20

<212>DNA

<213> ADAMTS1 Probe

<400>24

aaagaagcga tttgtgtcca 20

<210>25

<211>23

<212>DNA

<213> BACE2 Forward primer

<400>25

ggaatggaat acttggccta gct 23

<210>26

<211>21

<212>DNA

<213> BACE2 reverse primer

<400>26

caccagggag tcgaagaagg t 21

<210>27

<211>27

<212>DNA

<213> BACE2 Probe

<400>27

atgccacact tgccaagcca tcaagtt 27

<210>28

<211>26

<212>DNA

<213> forward primer of DSCR5

<400>28

gaatcttggc taaactcttt aggttt 26

<210>29

<211>20

<212>DNA

<213> DSCR5 reverse primer

<400>29

aggtaatgca actgcccaat 20

<210>30

<211>18

<212>DNA

<213> DSCR5 Probe

<400>30

acctattggc ctcaaaaa 18

<210>31

<211>17

<212>DNA

<213> ITSN1 Forward primer

<400>31

tggtggcagc ctggata 17

<210>32

<211>20

<212>DNA

<213> ITSN1 reverse primer

<400>32

atcatgcttc gctctttcct 20

<210>33

<211>14

<212>DNA

<213> ITSN1 Probe

<400>33

ctgggccata actg 14

<210>34

<211>20

<212>DNA

<213> PLAC4 Forward primer

<400>34

cctttccccc ttatccaact 20

<210>35

<211>22

<212>DNA

<213> PLAC4 reverse primer

<400>35

gtactggttg ggctcatttt ct 22

<210>36

<211>15

<212>DNA

<213> PLAC4 Probe

<400>36

ccctagccta taccc 15

<210>37

<211>20

<212>DNA

<213> LOC90625 Forward primer

<400>37

tgcacatcgg tcactgatct 20

<210>38

<211>20

<212>DNA

<213> LOC90625 reverse primer

<400>38

ggtcagtttg gccgataaac 20

<210>39

<211>16

<212>DNA

<213> LOC90625 Probe

<400>39

cctactggca cagacg 16

<210>40

<211>20

<212>DNA

<213> RPL17 Forward primer

<400>40

tgagggttga ctggattggt 20

<210>41

<211>21

<212>DNA

<213> RPL17 reverse primer

<400>41

tacagcactg cttccacaga a 21

<210>42

<211>14

<212>DNA

<213> RPL17 Probe

<400>42

aggcccgtgt ggct 14

<210>43

<211>90

<212>DNA

<213> COL6A1 calibrator sequence

<400>43

tggacaaagt caagtccttc accaagcgct tcatcgacaa cctgagggac aggtactacc 60

gctgtgaccg aaacctggtg tggaacgcag 90

<210>44

<211>61

<212>DNA

<213> COL6A2 calibrator sequence

<400>44

gagatcaacc aggacaccat caaccgcatc atcaaggtca tgaaacacga agcctacgga 60

g 61

<210>45

<211>65

<212>DNA

<213> ATP50 calibrator sequence

<400>45

tcccctcact accaacctga tcaatttgct tgctgaaaat ggtcgattaa gcaataccca 60

aggag 65

<210>46

<211>65

<212>DNA

<213> SOD1 calibrator sequence

<400>46

tgcagggcat catcaatttc gagcagaagg aaagtaatgg accagtgaag gtgtggggaa 60

gcatt 65

<210>47

<211>18

<212>DNA

<213> TFRC Forward primer

<400>47

cggctgcagg ttcttctg 18

<210>48

<211>25

<212>DNA

<213> TFRC reverse primer

<400>48

gttagagaat gctgatctag cttga 25

<210>49

<211>16

<212>DNA

<213> TFRC Probe

<400>49

tggcagttca gaatga 16

<210>50

<211>20

<212>DNA

<213> forward primer for EFEMP1

<400>50

cacaacgtgt gccaagacat 20

<210>51

<211>22

<212>DNA

<213> EFEMP1 reverse primer

<400>51

cgtaaattga tgcacacttg gt 22

<210>52

<211>18

<212>DNA

<213> EFEMP1 Probe

<400>52

acgcacaact gtagagca 18

<210>53

<211>21

<212>DNA

<213> ATP50 Forward primer

<400>53

ccctcactac caacctgatc a 21

<210>54

<211>21

<212>DNA

<213> ATP50 reverse primer

<400>54

ccttgggtat tgcttaatcg a 21

<210>55

<211>15

<212>DNA

<213> ATP50 Probe

<400>55

tgcttgctga aaatg 15

<210>56

<211>18

<212>DNA

<213> IGFBP3 Forward primer

<400>56

agtccaagcg ggagacag 18

<210>57

<211>22

<212>DNA

<213> IGFBP3 reverse primer

<400>57

caggtgattc agtgtgtctt cc 22

<210>58

<211>16

<212>DNA

<213> IGFBP3 Probe

<400>58

aatatggtcc ctgccg 16

<210>59

<211>18

<212>DNA

<213> ABP1 Forward primer

<400>59

tggaagcaga gcgaactg 18

<210>60

<211>17

<212>DNA

<213> ABP1 reverse primer

<400>60

catcaggatg gcagcca 17

<210>61

<211>13

<212>DNA

<213> ABP1 Probe

<400>61

agcgagagat gcc 13

<210>62

<211>18

<212>DNA

<213> FN1 Forward primer

<400>62

aagcaagccc ggttgtta 18

<210>63

<211>19

<212>DNA

<213> FN1 reverse primer

<400>63

ccaacgcatt gcctaggta 19

<210>64

<211>20

<212>DNA

<213> FN1 Probe

<400>64

acactatcag ataaatcaac 20

<210>65

<211>16

<212>DNA

<213> INHBA Forward primer

<400>65

cgccctccca aaggat 16

<210>66

<211>23

<212>DNA

<213> INHBA reverse primer

<400>66

gcatgtttaa aatgtgcttc ttg 23

<210>67

<211>26

<212>DNA

<213> INHBA Probe

<400>67

tacccaactc tcagccagag atggtg 26

<210>68

<211>19

<212>DNA

<213> SLC21A2 Forward primer

<400>68

gctttgggct ctccagttc 19

<210>69

<211>24

<212>DNA

<213> SLC21A2 reverse primer

<400>69

gtagctgaca aagatgatga ggat 24

<210>70

<211>18

<212>DNA

<213> SLC21A2 Probe

<400>70

tttccagctt gaatgaga 18

<210>71

<211>17

<212>DNA

<213> SIGLEC6 Forward primer

<400>71

caagctctct gtgcgtg 17

<210>72

<211>19

<212>DNA

<213> SIGLEC6 reverse primer

<400>72

gtccctggga tggagatgt 19

<210>73

<211>14

<212>DNA

<213> SIGLEC6 Probe

<400>73

atggccctga ccca 14

<210>74

<211>20

<212>DNA

<213> KIAA0992 Forward primer

<400>74

acctgtttgg ctacgaatcc 20

<210>75

<211>22

<212>DNA

<213> KIAA0992 reverse primer

<400>75

gaatctgttg aactggcacc tt 22

<210>76

<211>17

<212>DNA

<213> KIAA0992 Probe

<400>76

acatctgctg aggtgtt 17

<210>77

<211>19

<212>DNA

<213> TIMP3 Forward primer

<400>77

ccttctgcaa ctccgacat 19

<210>78

<211>18

<212>DNA

<213> TIMP3 reverse primer

<400>78

agcttcttcc ccaccacc 18

<210>79

<211>14

<212>DNA

<213> TIMP3 Probe

<400>79

cgtgatccgg gcca 14

<210>80

<211>20

<212>DNA

<213> LEP Forward primer

<400>80

ggtgagagct gctctggaaa 20

<210>81

<211>21

<212>DNA

<213> LEP reverse primer

<400>81

cctcagcctg attaggtggt t 21

<210>82

<211>14

<212>DNA

<213> LEP Probe

<400>82

tgacccagat cctc 14

<210>83

<211>21

<212>DNA

<213> LPL Forward primer

<400>83

agcaaaacct tcatggtgat c 21

<210>84

<211>22

<212>DNA

<213> LPL reverse primer

<400>84

gcacccaact ctcatacatt cc 22

<210>85

<211>15

<212>DNA

<213> LPL Probe

<400>85

tggctggacg gtaac 15

<210>86

<211>20

<212>DNA

<213> PLAC4 Forward primer

<400>86

cctttccccc ttatccaact 20

<210>87

<211>22

<212>DNA

<213> PLAC4 reverse primer

<400>87

gtactggttg ggctcatttt ct 22

<210>88

<211>15

<212>DNA

<213> PLAC4 Probe

<400>88

ccctagccta taccc 15

<210>89

<211>74

<212>DNA

<213> PLAC4 calibrator

<400>89

cacctttccc ccttatccaa ctagccctag cctataccct ctgctgccca agaaaatgag 60

cccaaccagt acac 74

<210>90

<211>18

<212>DNA

<213> FN1 Forward primer

<400>90

aagcaagccc ggttgtta 18

<210>91

<211>19

<212>DNA

<213> FN1 reverse primer

<400>91

ccaacgcatt gcctaggta 19

<210>92

<211>20

<212>DNA

<213> FN1 Probe

<400>92

acactatcag ataaatcaac 20

<210>93

<211>85

<212>DNA

<213> FN1 calibrator

<400>93

aaagcaagcc cggttgttat gacaatggaa aacactatca gataaatcaa cagtgggagc 60

ggacctacct aggcaatgcg ttggt 85

<210>94

<211>16

<212>DNA

<213> INHBA Forward primer

<400>94

cgccctccca aaggat 16

<210>95

<211>23

<212>DNA

<213> INHBA reverse primer

<400>95

gcatgtttaa aatgtgcttc ttg 23

<210>96

<211>26

<212>DNA

<213> INHBA Probe

<400>96

tacccaactc tcagccagag atggtg 26

<210>97

<211>76

<212>DNA

<213> INHBA calibrator

<400>97

ccgccctccc aaaggatgta cccaactctc agccagagat ggtggaggcc gtcaagaagc 60

acattttaaa catgct 76

<210>98

<211>20

<212>DNA

<213> LEP Forward primer

<400>98

ggtgagagct gctctggaaa 20

<210>99

<211>21

<212>DNA

<213> LEP reverse primer

<400>99

cctcagcctg attaggtggt t 21

<210>100

<211>14

<212>DNA

<213> LEP Probe

<400>100

tgacccagat cctc 14

<210>101

<211>62

<212>DNA

<213> LEP calibrator

<400>101

gggtgagagc tgctctggaa aatgtgaccc agatcctcac aaccacctaa tcaggctgag 60

gt 62

<210>102

<211>24

<212>DNA

<213> ADAM12 Forward primer

<400>102

tggaaagaaa tgaaggtctc attg 24

<210>103

<211>19

<212>DNA

<213> ADAM12 reverse primer

<400>103

tcgagcgagg gagacatca 19

<210>104

<211>29

<212>DNA

<213> ADAM12 Probe

<400>104

cacggaaacc cactatctgc aagacggta 29

<210>105

<211>84

<212>DNA

<213> ADAM12 calibrator

<400>105

tggaaagaaa tgaaggtctc attgccagca gtttcacgga aacccactat ctgcaagacg 60

gtactgatgt ctccctcgct cgaa 84

<210>106

<211>21

<212>DNA

<213> PAPPA2 Forward primer

<400>106

cacagtggaa gcctgggtta a 21

<210>107

<211>22

<212>DNA

<213> PAPPA2 reverse primer

<400>107

atcaaacaca cctgcgatga tg 22

<210>108

<211>24

<212>DNA

<213> PAPPA2 Probe

<400>108

ccggagggag gacagaacaa ccca 24

<210>109

<211>72

<212>DNA

<213> PAPPA2 calibrant

<400>109

tcacagtgga agcctgggtt aaaccggagg gaggacagaa caacccagcc atcatcgcag 60

gtgtgtttga ta 72

<210>110

<211>19

<212>DNA

<213> PAPPA Forward primer

<400>110

gggcattcac accatcagt 19

<210>111

<211>21

<212>DNA

<213> PAPPA reverse primer

<400>111

tcggtctgtc ttcaaggaga a 21

<210>112

<211>27

<212>DNA

<213> PAPPA Probe

<400>112

ccaagacaac aaagacccac gctactt 27

<210>113

<211>72

<212>DNA

<213> PAPPA calibrator

<400>113

tgggcattca caccatcagt gaccaagaca acaaagaccc acgctacttt ttctccttga 60

agacagaccg ag 72

Claims (10)

1. A kit for determining the presence of a fetus having trisomy 21 in a pregnant woman, the kit comprising:

(i) PCR primers for quantitative determination of the mRNA content in a biological sample of said pregnant woman, wherein said mRNA is an mRNA derived from the PLAC4 genetic locus, wherein said biological sample is blood, washes of the reproductive tract, amniotic fluid, urine, saliva or chorionic villi; and

(ii) a standard control representing the amount of said RNA type in a corresponding sample from a pregnant woman carrying a normal chromosomal fetus.

2. The kit of claim 1, wherein an increase in said mRNA level relative to said standard control indicates that said pregnant infant has a high susceptibility to trisomy 21.

3. The kit of claim 1, wherein the PCR comprises reverse transcription polymerase chain reaction.

4. The kit of claim 1, further comprising reagents for mass spectrometry.

5. The kit of any one of claims 1-4, wherein the pregnant woman is in the first, middle or last trimester of pregnancy.

6. The kit of any one of claims 1-4, wherein the blood is plasma or serum.

7. The kit of any one of claims 1-4, wherein the mRNA level is increased more than 2-fold over a standard control.

8. The kit of claim 5, wherein the pregnant woman is in the first, middle or last trimester of pregnancy.

9. The kit of claim 5, wherein the mRNA level is increased more than 2-fold over a standard control.

10. The kit of claim 6, wherein the mRNA level is increased more than 2-fold over a standard control.