WO2006069592A2

WO2006069592A2 - Method for diagnosing an/or predicting preeclampsia and/or related disorders

Info

Publication number: WO2006069592A2
Application number: PCT/EP2004/014879
Authority: WO
Inventors: Cornelis Bartholomeus Maria Oudejans; Marie Van Dijk; Joyce Mulders
Original assignee: Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patientenzorg
Priority date: 2004-12-31
Filing date: 2004-12-31
Publication date: 2006-07-06
Also published as: WO2006069592A3

Abstract

The present invention relates to methods for predicting and/or diagnosing a pregnancy at risk for preeclampsia and/or related disorders, comprising: (a) obtaining at least one marker nucleic acid from a biological sample; (b) providing the diagnosis based on at least one of the quantity, activity and/or sequence of said marker nucleic acid in said sample. The invention further relates to the marker nucleic acid per se for use in predicting, diagnosing, preventing and/or treating preeclampsia and/or related disorders, as well as to kits and primers for use in the methods of the invention.

Description

METHOD FOR DIAGNOSING AND/OR PREDICTING PREECLAMPSIA AND/OR RELATED DISORDERS

Field of the invention The present invention relates to a method for diagnosing and/or predicting a pregnancy at risk for preeclampsia and/or related disorders with placenta dysfunction. The present invention further relates to kits and primers for performing said methods, as well as marker nucleic acids for use in the diagnosis.

Background of the invention

Preeclampsia, commonly referred to as gestational hypertension with proteinuria, is the most frequent pregnancy-associated disorder and is the leading cause of maternal and fetal morbidity and mortality. Preeclampsia starts with placental dysfunction in the first trimester followed by maternal systemic (de) compensation, mainly characterized by endothelial dysfunction with clinical symptoms presenting in the second and third trimester of pregnancy. Therefore, although symptoms are late and maternal, the origin of preeclampsia is early and placental.

Currently, testing for this disorder involves the use of serum or plasma markers, such as hepatocyte growth factor (HGF) , C-reactive protein (CRP) , placenta growth factor

(PGF) , and E-selectin, that lack sufficient discrimination or focus erroneously on secondary maternal symptoms. Truly discriminative methods for early, i.e. presymptomatic detection, and as such for primary and secondary prevention of preeclampsia, are greatly hampered by lack of insight in the placental etiology of preeclampsia. Description of the invention

It is an object of the present invention to provide novel diagnostic methods for the presymptomatic detection and/or prediction of a pregnancy at risk for preeclampsia and/or related disorders with placenta dysfunction.

This is achieved by the invention by providing a method, comprising:

(a) detecting at least one marker nucleic acid in a biological sample; (b) providing a diagnosis based on at least one of the quantity, activity and/or sequence of said marker nucleic acid.

In a first aspect the present invention thus relates to a method for performing a prenatal and presymptomatic diagnosis and/or prediction of a pregnancy at risk for preeclampsia. This method comprises (a) detecting at least one marker nucleic acid in a biological sample and (b) providing the diagnosis based on the quantity, activity and/or sequence of said marker nucleic acid. A marker nucleic acid in this respect relates to a nucleic acid, i.e. RNA, or cDNA derived from said RNA or DNA, obtained from a biological sample, of which at least one of the quantity, activity and/or sequence is indicative of preeclampsia.

Step (a) of the method usually will involve (1) the extraction of the marker nucleic acid, for example placenta- derived or associated RNA or DNA, from the biological sample;

(2) the application of some form of a nucleic acid amplification assay to amplify the extracted RNA or DNA, whereby in the case of RNA this is first to be reverse transcribed to cDNA prior to amplification of the cDNA; and

(3) the detection of at least one marker RNA or DNA or their amplification products. The amplification and detection steps (2) and (3) may be performed so as to allow either qualitative or quantitative detection of the marker nucleic acid, depending upon the ultimate clinical relevance of the marker nucleic acid in question with respect to establishing the diagnosis of a pregnancy at risk for preeclampsia. In a particularly preferred embodiment of the present invention, providing the diagnosis is based on the sequence of the marker nucleic acid and comprises detecting at least one mutation and/or modification in said marker nucleic acid. Following the extraction of said marker nucleic acid from the biological sample, and determining the sequence thereof, one or more specific mutations or genetic modifications, such as methylation of the gene, may thus be detected therein in a qualitative and/or quantitative manner so to allow the diagnosis or prediction of a pregnancy at risk for preeclampsia.

Preferably, the marker nucleic acid is a placental marker nucleic acid, i.e. the marker nucleic acid preferably is derived from a placental gene, e.g. an RNA which is transcribed from said placental gene, or the cDNA derived therefrom. The placental marker nucleic acid may also be at least part of said placental gene. It is thus to be understood that according tot the present invention the term marker nucleic acid relates to all possible forms of nucleic acid, i.e. RNA and DNA, including messenger RNA and cDNA. In the research that led to the present invention, a linkage of chromosome 10q22 with preeclampsia in the Netherlands was demonstrated. Within a 444 kb maternal effect region, that resides in a female-specific recombination hotspot, a gene, Cl0orf24, was identified with missense mutations present in all patients and which were identical between sisters of the same family. It was furthermore demonstrated that the protein encoded by this gene belongs to a novel family of winged helix domain containing transcription factors, related to, but distinct from the Forkhead (FOX) family. This novel gene was tentatively called Storkhead 1 (STOXl) . Placental expression of this gene was shown to include the cells primarily affected in preeclampsia, i.e. the invasive extravillus trophoblast cells . The mutations found in this gene show transmission distortion with matrilineal inheritance. One mutation, Y153H, was found in almost all preeclamptic females (46 out of 48 patients tested) . This change represents a non-conservative mutation. That is, all FOX proteins (n=38) and all C10orf24 homologs (n=8) have either a tyrosine or a phenyl alanine at its corresponding position in the winged helix domain. In addition, nucleotide changes leading to substitution of amino acids at other sited in the protein can cause disease: the F112S mutation in FOXCl for example leads to glaucoma. This gene thus may be responsible for preeclampsia in the Netherlands. A paralog of this gene (DKFZp762K222; Accession NO: NM_020225) is located on 4q35 in a chromosomal region linked with preeclampsia in another population (Australia/New Zealand) . This indicates the existence of founder dependent mutations in paralogous genes sharing the same pathway and being dysfunctional in preeclampsia. This pathway is related to the induction of polyploidization of trophoblast cells where differentiation of invasive trophoblast to non-invasive trophoblast cells is accompanied by endoreduplication.

Endoreduplication is an unusual form of cell cycle in which rounds of DNA synthesis repeat in the absence of mitosis, thus leading to the formation of differentiated polyploid cells. Besides trophoblast cells, endoreduplication is found in megakaryotes, liver cells and cardiomyocytes .

The marker nucleic acid according to the present invention thus preferably is derived from, a gene from the 10q22 region, selected from the group consisting of MAWBP (Accession NO: NM_022129), HNRPH3 (Accession NO: NM__012207), RUFY2 (Accession NO: NM_017987), DNA2L (Accession NO: XM_166103), SLC25A16 (Accession NO: 152707), CXXC6 (Accession NO: NM_030625), CCARl (Accession NO: NM_018237) , C10orf24 (figure 5), and DDX50 (Accession NO: NM_024045) . The skilled person will understand that likewise the sequences complementary to these sequences and/or allelic variants or their complements, or fragments thereof may be used.

In a particular preferred embodiment, the marker nucleic acid according to the invention is a nucleic acid derived from the gene C10orf24, comprising a nucleotide sequence as represented by SEQ ID. No. 1 (Figure 5) . This gene, which is located on human chromosome 10q22, as described above, has been demonstrated to be linked to preeclampsia in the research that led to the present invention, and as such is highly suitable for diagnosing a pregnancy at risk for preeclampsia. It should be understood, however, that also other paralogs, i.e. genes from the same homologous superfamily found in another part of the human genome, such as DKFZp762K222 (Accession NO: NM_020225) , and marker nucleic acids derived therefrom, may be used in the method of the present invention.

Preferably, the marker nucleic acid is derived from C10orf24.1, C10orf24.2, C10orf24.3 and/oror C10orf24.4 (Accession Nos : AY842014, AY842015, AY842016, AY842017, respectively), represented in Figure 5-9.

The present invention also relates to these placental marker nucleic acids, per se, as well as to the placental gene C10orf24 for use in predicting, diagnosing, preventing and/or treating preeclampsia and/or related disorders with placenta dysfunction characterized by disturbances of trophoblast polyploidy. According to the present invention it has been found that a pregnancy is at risk for preeclampsia if one or more of the following mutations are detected in the sequence of cDNA derived from a maternal blood sample (exon, amino acid change, position in contig, nucleotide change) : a. Exon 1 R18P 19138588 G to C b. Exon 3 Y153H 19193015 T to C c. Exon 3 E608D 19196531 A to C d. Exon 3 N825I 19197181 A to T In this particularly preferred embodiment of the method of the present invention, the placental marker nucleic acid thus preferably is derived from a foetal gene, and contains one or more mutations inherited from the mother. Identification of the specific mutation involved may for example be performed by prior DNA analysis of all coding sequences of the C10orf24 gene in DNA isolated from. e.g. the mother and/or the father using buccal swabs .

In another preferred embodiment of the present invention, providing the diagnosis comprises comparing the quantity of the marker nucleic acid, preferably the placental marker nucleic acid, in the biological sample to the quantity of a reference nucleic acid. In this way, the quantity of the marker nucleic acid in the sample may be corrected for biological and experimental variability. Preferably, the reference nucleic acid is a placental reference nucleic acid, e.g. an RNA derived from a different chromosome or chromosomal region than 10q22, or from the 10q22 region outside the minimal critical region between CCARl and RUFYl. More preferably, the placental reference nucleic acid is a nucleic acid that is of trophoblastic, in particular of extravillus origin, i.e. at least expressed in those types of placental cells. A preferred placental reference nucleic acid for quantification of the placental marker nucleic acid in the methods of the invention is a nucleic acid derived from a gene selected from a group of genes with related function, i.e. transcription factors. Preferably, the placental marker nucleic acid is a nucleic acid derived from the gene GCMl (Accession NO. NM_003643) , and/or a fragment, allelic variants and/or the complements thereof. In another preferred embodiment, the reference nucleic acid may be derived from an identical gene as the marker nucleic acid, but be obtained from persons not suffering from preeclampsia, i.e. healthy pregnant persons. A quantitative change (i.e. increase or decrease) of one or more of the marker nucleic acids, or of the unmethylated form of C10orf24 DNA in the maternal blood compared to the reference nucleic acid can thus identify a pregnancy at risk for developing preeclampsia.

In another preferred embodiment of the present invention, providing the diagnosis comprises determining the activity of the marker nucleic acid in the biological sample. Dysfunctioning of a gene or pathway involving said gene may result in quantitative changes of the placental marker nucleic acid, e.g. a decrease of placental mRNA or cDNA in the sample.

The number of copies of one or more placental marker nucleic acids RNAs may be determined in a biological sample, e.g. using a real time quantitative RT-PCR as described below, as well as the number of copies of one or more placental reference nucleic acids. Preferably, the average value of the number of copies of a given placental marker nucleic acid in a given quantity of a biological sample from healthy control pregnancies is determined for a given age of gestation by normalizing the value to the average number of copies of the one or more placental reference nucleic acids in the same biological samples. A preeclamptic pregnancy may then be diagnosed if the normalized number of copies of a given placental marker nucleic acid in the sample is at least 1.1, 1.2, 1.4, or 1.8 times higher or lower than the average value of that marker nucleic acid for the healthy control pregnancies of (about) the same age of gestation.

According to the invention, the biological sample preferably is a sample of a maternal bodily fluid, i.e. a biological sample derived from the mother. Preferably, the biological sample is blood sample obtained from a pregnant woman during early pregnancy, i.e. preferably the sample is obtained in the first trimester of the pregnancy, more preferably the sample is obtained at least prior to week 17, 16, 15, 14, 13, 12, 11, 10, 9 or 8 of gestation. Other types of biological samples which may be used in the method of the present invention include e.g. urine or amniotic fluid. However, the pre-screening of the mother alone, or both mother and father, before pregnancy in order to predict preeclampsia forms also part of the present invention. The methods according to the invention thus are preferably performed ex vivo on a blood sample that is obtained from a pregnant female. Either "fresh" blood, plasma or serum, or frozen (stored) and subsequently thawed plasma or serum may be used for purposes of this invention. Frozen (stored) plasma or serum should optimally be maintained at storage conditions of -20 to -70 degrees centigrade until thawed and used. "Fresh" plasma or serum should be refrigerated or maintained on ice until used, with RNA extraction being performed as soon as possible. Blood may be drawn by standard methods into a collection tube, preferably siliconized glass, either without anticoagulant for preparation of serum, or with EDTA, sodium citrate, heparin, or similar anticoagulants for preparation of plasma. The preferred method if preparing plasma or serum for storage, although not an absolute requirement, is that plasma or serum be first fractionated from whole blood prior to being frozen. This reduces the burden of extraneous intracellular RNA released from lysis of frozen and thawed cells which might reduce the sensitivity of the amplification assay or interfere with the amplification assay through release of inhibitors to PCR such as porphyrins and hematin. Thus, in a preferred method of the invention, all nucleated and anucleated cell populations are removed from the blood sample prior to detection of placental RNA or DNA. More preferably, the placental RNA or DNA is detected in maternal blood plasma or serum. "Fresh" plasma or serum may be fractionated from whole blood by centrifugation, using gentle centrifugation at 300-800 x g for five to ten minutes, or fractionated by other standard methods. Particularly preferred in the fractionation of plasma or serum from whole blood is the addition of a second centrifugation step for five to ten minutes at about 20.000 to 30.000 x g, more preferably at about 25.000 x g to improve the signal to noise ratio in subsequent RNA detection methods. Since heparin may interfere with RT-PCR, use of heparinized blood may require pretreatment with heparinase, followed by removal of calcium prior to reverse transcription, as described (Imai et al., 1992, J. Virol. Methods 36: 181-184) . In general, EDTA is the preferred anticoagulant for blood specimens in which PCR amplification is planned. In the methods of the invention, the placental marker RNA/DNA is usually detected in equal or less than 2 ml maternal blood, plasma or serum, more preferably in equal or less than 1.6, 0.8, 0.4, 0.2 or 0.1 ml of maternal blood, plasma or serum.

In the methods of the present invention, the marker nucleic acid may be extracted from the biological sample, preferably maternal bodily fluid, more preferably whole blood, and more preferably plasma or serum using e.g. nucleic acid extraction methods such as, but not limited to, gelatin extraction method; silica, glass bead, or diatom extraction method; guanidiniurα thiocyanate acid-phenol based extraction methods; guanidinium thiocyanate acid based extraction methods; guanidine-hydrochloride based extraction methods; methods using centrifugation through cesium chloride or similar gradients; phenol-chloroform based extraction methods; and/or other available nucleic acid extraction methods, as are known in the art for use in extraction of intracellular RNA/DNA, including commercially available RNA/DNA extraction methods, e.g. by using or adapting or modifying the methods of Chirgwin et al. (1979, Biochem. 18: 5294-5299), or WO97/35589.

Particularly preferred nucleic acid extraction methods for use in the methods of the invention are commercially available extraction methods suitable for extraction of intracellular and extracellular nucleic acids and in particular viral RNA, including e.g., TRIzol™ (Life Technologies); Trisolv™ (BioTecx Laboratories); ISOGEN™ (Nippon Gene) ; RNA Stat™ (Tel-test) ; TRI Reagent ™ (Sigma) ; SV Total RNA Isolation System (Promega) ; RNeasy Mini Kit, QIAamp MinElute Virus Spin or QIAamp MinELute Virus Vacuum Systems (Qiagen, Hilden, Germany) ; Perfect RNA: Total RNA

Isolation Kit (Five Prime-Three Prime Inc., Boulder, Colo.); or similar commercially available kit, wherein extraction of nucleic acids may be performed according to manufacturer's directions, adapted to the maternal blood, serum or plasma. Most preferably the QIAamp MinELute Virus Vacuum System is used as it reduces the presence of aspecific bands in RT-PCR.

Circulating extracellular DNA, as well as foetal- derived or associated extracellular DNA, is also present in maternal plasma and serum (see e.g. WO 98/39474) . Since this DNA will additionally be extracted during the RNA extraction methods described above, it may be desirable or necessary to further select for either placental RNA or DNA extract by pretreatment with either DNase or RNase, respectively, to permit selective analysis of either placental DNA, RNA or both. This may be accomplished using e.g. DNase in a method as described by Rashtchian (1994, PCR Methods Applic. 4: S83- S91) for analysis of RNA or accomplished using RNase for analysis of DNA.

In a preferred embodiment, the placental, marker nucleic acid is extracted from maternal blood, serum or plasma using one or more probes that specifically hybridize to specific nucleic acids. The probes may be attached to solid substrates or magnetic beads or particles, or may be probes whereby upon hybridization to a nucleic acid, an electrical gradient or magnetic gradient or density gradient enables extraction and/or separation of specific nucleic acid species from the remainder of bodily fluid. Further, the nucleic acid may be hybridized to a solid substrate at a bio- electrical interface whereupon hybridization of a specific RNA, or cDNA derived therefrom, generates an electrical signal which may further be amplified and detected.

For detection, reverse transcription, amplification and hybridization of placental marker nucleic acids in the methods of the invention unique primers and probes may be designed based on the available sequences of expressed placental RNA's in databases. A unique priming sequence preferably is a sequence that is suitable to serve as a primer-binding site for amplification primers in PCR. The length of the priming sequence may vary from 15 to 40, preferably from 18 to 30, more preferably from 20 to 25. A priming sequence preferably is optimized to meet a number of criteria for optimal use as PCR primer, such e.g. the absence of sequences that can form hairpins or other secondary structures. The priming sequence may bind only to a single site in the marker nucleic acid. It may therefore be useful to test that the selected primer sequence does not demonstrate significant matches to sequences in the GenBank database (or other available databases) . Furthermore, the Tm (also referred to as Tann) of the primer may be optimized by analysis of the length and GC content of the primer. Such optimal priming sequences can be designed using a standard PCR-primer selection program such as "Primer Designer" version 2.0 (copyright 1990, 1991, Scientific and Educational software), "PrimerSelect" of the DNAStar™ software package (DNAStar, Inc.; Madison, Wis.), and "Oligo 4.0" (National Biosciences, Inc.) . Preferred forward and reverse primers for use in the methods of the invention are identified in table 1 and 2. Particularly preferred primers for use in the method of the present invention are identified in table 1.13 and table 2. The present invention also relates to these primers per se.

As described above, the placental marker and reference nucleic acids extracted from the biological sample are preferably amplified in vitro. Applicable amplification assays include but are not limited to reverse transcriptase polymerase chain reaction (RT-PCR) , ligase chain reaction,

RNA and cDNA signal amplification methods including branched chain signal amplification, amplifiable RNA reporters, Q-beta replication, transcription-based amplification, boomerang DNA amplification, strand displacement activation, cycling probe technology, isothermal nucleic acid sequence based amplification (NASBA) , other self sustained sequence replication assays, and other nucleic acid amplification assays as known in the art, and/or any variations or combinations thereof, performed in either qualitative or quantitative fashion. For example, the methods of the invention can utilize nucleic acid amplification methods as known in the art, such as but not limited to adapting those described by Edmands et al. (1994, PCR Methods Applic. 3:

317-319); Abravaya et al. (1995, Nucleic Acids Res. 23: 675- 682); Urdea et al . (1993, AIDS 7 (suppl 2) : S11-S14) ; Kievits et al. (1991, J. Virological Methods 35: 273-286); and in WO97/35589. In preferred embodiments of the methods of the invention, placental RNA is converted into cDNA using reverse transcriptase prior to in vitro amplification using methods known in the art. For example, a sample, such as 10 μl extracted plasma or serum RNA is reverse transcribed in a 30 μl volume containing 200 Units of Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Madison, Wis.), a reaction buffer supplied by the manufacturer, 1 mM dNTPs, 0.5 micrograms random hexamers, and 25 Units of RNAsin (Promega, Madison, Wis.) . Reverse transcription is typically performed under an overlaid mineral oil layer to inhibit evaporation and incubated at room temperature for 10 minutes followed by incubation at 37⁰C for one hour. Alternatively, other methods well known in the art can be used to reverse transcribe the mammalian RNA to cDNA. There are numerous methods available in the art for the detection of nucleic acids, any of which may be used in the methods of the invention for the qualitative or quantitative detection of the placental marker nucleic acid A amplified as described above. A preferred method uses gel electrophoresis, such as e.g. electrophoresis in agarose or polyacrylarαide gels (see e.g. in Sambrook and Russel, 2001, In: "Molecular Cloning: A Laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, NY) . As an alternative to ethidium bromide or other in gel detection methods, the amplified product can be transferred from the gel to a membrane by blotting techniques to be detected with a labeled probe. Amplified products may also be detected using immunological detection methods such as e.g. described by Landgraf et al. (1991, Anal. Biochera. 198: 86-91; 1991, Anal. Biochem. 193: 231-235), Coutlee et al. (1989, Anal. Biochem. 181: 96-105) and Bobo et al. (1990, J. din Micra 28: 1968-1973) or electrochemiluminescence detection methods, such as described by Blackburn et al .

(1991, Olin. Chem. 37: 1534-1539), or DiCesare et al. (1993, BioTechniques 15: 152-157), all methods utilizing reverse dot blot detection technology (Saiki et al., 1989, Science 233: 1076-1078), and all methods utilizing high-performance liquid chromatography.

For quantitative detection of the amplified products in the methods of the present invention, real time PCR may be used. Real time PCR amplification allows the quantitative detection of the logarithmically increasing amount of PCR product in a specific PCR reaction. Three main real-time PCR machines are currently on the market: (1) The light cycler, developed by ROCHE

(http://www.biochem.roche.com/liqhtcycler/) (Wittwer et al. 1989, Nucleic Acids Res. 17: 4353-7; Wittwer et al. , 1997, Biotechniques . 22: 176-81), (2) the Taqman (commercialized by Perkin Elmer-Applied Biosystems

(http: //www.appliβdbiosysterns.com/products/, generating information on the ABI-PRISM 7700, 7900HT, and 5700 machines) , and the (3) iCycler commercialised by BIO-RAD (http://www.bio-rad.com/iCyclβr/) (for an overview of characteristics of the respective machines, the reader is referred to a molecular biology tools website: www.nlv.ch/Molbioltoolsrtpcr.html. (See also Bustin, 2000, J. MoI. Endocrinol. 25: 169-93) . All three technologies depend on a similar detection method that is based on the real-time detection during a PCR amplification, of a fluorescent signal, the strength of which is proportional to the specific PCR product that is amplified. Yet, the molecular basis that underlies the generation of a quantitative fluorescence signal that corresponds to the amount of the PCR product is different in these three technologies. The Light Cycler e.g., can be used with a double strand DNA (dsDNA) fluorophore that specifically interacts with ds-DNA but does not produce a fluorescent signal with single strand DNA. Thus with increasing amounts of ds-DNA, generated through PCR amplification of the template, an increasing level of fluorescence is generated, thereby allowing quantification. A disadvantage of this method is that the generation of the fluorescent signal does not involve any specificity for the nucleotide sequence that is amplified. As a consequence, any dsDNA molecule in the reaction mixture, including aspecific amplification products, will contribute to the signal, which will result in an overestimation of the specific amplification product.

Other real-time detection techniques do depend on nucleotide sequence specific fluorescence. For the Light Cycler, a hybridization probe detection system has been set- up that utilizes fluorophore energy transfer between two fluorescent groups. Similarly, detection tools have been developed for the Taqman and iCycler machines: SYBR® green (Morrison et al . , 1998, Biotechniques 24: 954-8, 960, 962), TaqMan® probes (inter alia DNA-binding dyes, molecular beacons, hydrolysis probes), Molecular Beacons® (Stratagene) (Tyagi and Kramer, 1996, Nat. Biotechnol. 14: 303-8), and others, that are based on variable physical characteristics of the compounds used and generate a quantitative fluorescence signal reflecting the logarithmically increasing DNA duplex molecule during the PCR reaction cycles that is either non-specific or sequence specific.

In a further aspect, the present invention relates to kits for. prenatal diagnosis and/or prediction of preeclamptic pregnancies, based on the methods as described above. Said kits may comprise a carrier to receive therein one or more containers, such as tubes or vials. The kit may further comprise unlabeled or labeled oligonucleotides (primers and/or probes) of the invention, which may be contained in one or more of the containers. The oligonucleotides may be present in lyophilized form, or in an appropriate buffer. One or more enzymes or reagents for use in reverse transcription and/or amplification reactions may further be contained in one or more of the containers. The enzymes or reagents may be present alone or in admixture, and in lyophilized form or in appropriate buffers. The kit may also contain any other component necessary for carrying out the methods of the present invention, such as buffers, enzymes, pipettes, plates, nucleic acids, nucleoside triphosphates, and gel materials. Such other components for the kits of the invention are known per se.

The kits preferably at least comprises primers or probes for detection and/or amplification of a marker nucleic acid, as described above.

In another preferred embodiment of the invention, the kit further comprises primers or probes used for the detection of an extracted placental reference nucleic acid, which hybridize to a nucleic acid derived from the gene GCMl (Accession NO: NM_003643) .

The present invention is further illustrated by the following figures and examples, which, however, are not to be construed as limiting. The features disclosed in the foregoing description, in the following figures, examples and in the claims may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Description of the figures

Fig. 1. Minimal critical region on 10q22 with maximal sharing of both alleles in affected sisters with preeclampsia.

The region with maximal sharing of both paternal and maternal alleles between affected sisters with preeclampsia of the same family is colored grey. This minimal critical region is restricted to a 444 kb fragment bordered by RUFY2 and CCARl and resides within the female-specific recombination hotspot recently identified between D10S599- D10S6767. GATA121A08 indicates the position of one of the markers previously shown to have genome wide linkage with preeclampsia. SNPs were identified by sequencing the PCR amplified exons of the 17 genes indicated. About 82,000 bp were sequenced in each patient with an average informativity (percentage of coding sequences analyzed of the indicated genes) of 76%. The percentages for each individual gene as well as the PCR conditions and primer sequences can be found in Table 1. Positions of SNPs correspond to positions in contig NT__008583_16. For clarity, annotations correspond to the same strand. SNPs leading to amino acid substitutions are indicated in black. Dark grey boxes indicate absence of allele sharing. Affected sibs (sisters) (indicated by 0, 1 and 4) had preeclamptic pregnancies, except 9506/1 having eclampsia. Their mothers had either preeclampsia (families 9267, 9268, 9510, 9526) or pregnancy-induced hypertension (families 9046, 9223, 9506, 9514) . Fig. 2. Features of genes in the minimal critical region of preeclampsia.

The majority of genes (A) within the minimal critical region (RUFY2-CCAR1) code for DNA-binding proteins (B) . Flanking genes (BNRPH3, DDX50) code for RNA-binding proteins. All genes are expressed in normal first trimester placentas (C) . Two genes, MAWBP and C10orf24, show downregulated expression in androgenetic placentas (complete hydatidiform moles) of identical gestational age (D) .

Fig. 3. Transcriptional organization and expression pattern of C10orf24 in early placenta. a. By differential donor splice site use, exon 3 skipping and alternative use of exon 5, six different transcripts are generated (A-F) coding for three different proteins. Identical colors in exons indicate identical protein sequences. Arrows indicate positions of primers used for transcript specific analysis. Exon 1 contains the bihelical nuclear localization signal. Exon 2 and first part of exon 3 contain the DNA binding winged helix domain (see also Table 1) . The second part of exon 3 contains the nuclear export sequence; b. Isoforms A-D are expressed in the early placenta. CB: total placenta, Vc: villus fibroblast cells from stroma of chorionic villi, EVT: SGHPL5 extravillus trophoblast cells; c. Nuclear expression of isoform A in extravillus trophoblast is restricted to polyploid cells, cytoplasmic expression to diploid cells. Nuclear and cytoplasmic expression is exclusive. Isoforms B and C are localized in the nuclei only. Isoform C lacking part of the winged helix domain but retaining the nuclear localization domain is localized in nucleoli. Fig. 4. Matrilineal transmission of heterozygous missense mutations in preeclampsia.

Pedigree structure of 3 generations of Dutch preeclampsia families (n=8) with linkage to chromosome 10q22. Females in generation I had either pregnancy-induced hypertension (grey circle) or preeclampsia (dark circle) . All affected sisters in generation II had preeclampsia with exception of 9506/1 who had eclampsia (dark stippled circle) . Generation III consists of children born from preeclamptic pregnancies (III) (shaded, PE) or born from normal pregnancies (white, N) . The mutant alleles (indicated in black) are always identical between affected sibs (generation II) of the same family and are only seen for the mutant alleles of the C10orf24 gene. This is not seen in the genes immediately flanking the C10orf24 gene. The C10orf24 mutations in generation II have been derived from the mother (generation I) as can be seen in 9046/1, 9223/1, 9268/0 and 9268/1. This feature correlates with the maternal effect seen in the linkage analysis. The mutations in generation II are transmitted to the children born from preeclamptic pregnancies (PE) with the maternal mutant allele always carried by the child born from the preeclamptic pregnancy with one exception (arrow) .

Fig. 5. Nucleotide sequence of the C10orf24 gene.

Fig. 6-9. Nucleotide sequence of C10orf24.1, C10orf24.2, C10orf24.3, Cl0orf24.4 respectively.

Table 1. Primers and PCR characteristics used for coding sequence analysis of 17 genes in the 10q22 region of preeclamptic females . Table 2. Primer and PCR characteristics used for transcipt analysis and cloning of C10orf24.

Table 3. Alignment of FOX proteins with the C10orf24 protein and its homologs.

A. Alignment of the winged helix domain of human FOX proteins indicates the conservation of hydrophic amino acids (marked in grey) that control wing 2 stability and as such control DNA binding specificity. In addition, all FOX proteins follow an absolutely conserved Y/F Y/F-6-W-7-L-4-F rule (marked in black) . In FOXO proteins, this rule is Y/F Y/F-11-W-7-L-4-F (not shown) . B. The C10orf24 protein (Human_A) has a similar secondary structure characteristic of a winged helix domain including the conservation of these hydrophobic amino acids. The YF rule specific for FOX proteins is absent in C10orf24 and its homologues indicating that this gene represents a novel member of the winged helix family. The human paralog (DKFZp762K222) on 4q35 is indicated by Human_B. The position of the Y153H mutation involving replacement of an absolutely conserved amino acid (Y or F) and found in almost all preeclamptic females is indicated by an arrow.

EXAMPLE

Maternal segregation of the Dutch preeclampsia locus at 10q22 with a novel member of the winged helix gene family

MATERIALS AND METHODS Patients. Families with two or more affected sib pairs (sisters) with preeclampsia were identified in the databases from 22 hospitals in the Netherlands and selected as described (Lachmeijer A.M. et al. , Eur. J. Hum. Gen. 9: 758-764 (2001) . Diagnostic criteria for gestational hypertension with or without proteinuria were: preeclampsia (PE) : de novo hypertension (diastolic BP ≥ 90 mm Hg with increment > 20 mm Hg from first trimester diastolic BP) and proteinuria > 300 mg/24h or at least twice 1+ on semiquantitative analysis; eclampsia (E) : seizures in hypertensive pregnancy with or without proteinuria; HELLP- syndrome: LDH ≥ 600 IU/L, ASAT and ALAT at least 70 IU/L and < 100 platelets x 109/L; and pregnancy-induced hypertension (PIH) : de novo hypertension in^' pregnancy without proteinuria. Genomic DNA was isolated from both parents (generation I) and the affected daughters (generation II) . From 8 families with preeclampsia, genomic DNA was obtained from 12 children (generation III) born from preeclamptic pregnancies and from 3 children born from normal pregnancies. Genomic DNA from children was obtained using buccal swaps followed by isolation using Qiagen kits. Genomic DNA from Kaukasian controls consisting of females (n=32) with no history of preeclampsia was isolated identically. All DNA was obtained following informed consent with permission of the Ethical Committees of the hospitals involved.

DNA and RNA analysis . Genomic DNA containing gene sequences were amplified by standard PCR methods in the presence of 1 M betaine. The primer sequences and PCR conditions are described, in Table 1. For sequencing, PCR fragments were following purification (Qiagen) subjected to cycle sequencing using Big Dye terminators and analyzed by capillary electrophoresis using a Genetic Analyzer 3100 (Applied Biosystems) . RNA and DNA from normal and androgenetic placentas (first trimester) as well as villus stromal cells (fibroblast) and extravillus trophoblast were obtained as described (Oudejans C.B.M. et al. , MoI. Hum. Reprod. 2004, van Dijk M. et al. , Gene Expression Patterns 5: 61-65, 2003) . First strand cDNA synthesis of the C10orf24 transcripts was performed with exon specific reverse primers overlapping the stop codon using Superscript II reverse transcriptase (InVitroGen) in a two-step, one-tube procedure. To permit specific reverse transcription across the high percentage GC first exon, RNA was preheated at 95°C for 1 rain, followed by cooling on ice, reverse transcription was started and performed at 55 °C in the presence of reverse primer only, with all steps performed in the presence of 1 M betaine. Following reverse transcription for 30 min, and heat inactivation for 1 min at 95^°C, the forward primer was added followed by PCR for 35 cycles (95°C, 1 min, 55-62^°C for 1 min, 72°C for 2 min) . For transcripts larger than 1200 bp and transcripts used for cloning RT-PCR was done with replacement of Taq polymerase by High Fidelity Pfu polymerase (InVitroGen) optimized for long templates with extension times of 3 min. Primer sequences and PCR conditions used for transcript analysis are described in Table 2.

Protein analysis. For subcloning in eGFP-Cl and -C2 vectors, reverse primers were flanked by a BamHI site, and forward primers with either EcoRI or BspEI sites and with replacement of the ATG initiation codon by ACG. Following digestion and purification, the cDNA fragments of 2970, 811 and 611 bp coding for isoforms A, B and C, respectively, were directionally cloned into pEGFP-Cl- (2970) or C2 (811, and 611) vectors (Clontech) . Transfection into SGHPL5, consisting of normal diploid extravillus trophoblast cells immortalized by SV40 transformation 28, was done as described using lipofection with FuGene (Roche) (Westerman B.A. et al. , Biochim. Biophys. Acta, 1676: 96-103, 2004) . Genbank accession numbers: Isoforrαs A-D of C10orf24: AY842014, AY842015, AY842016, AY842017.

URL addresses:.

SIFT v.2 program: http//blocks.fhcrc.org/sift/sift.html Psipred : http://bioinf-cs.ucl.ac.uk/psipred/psiforia.html MotifScan: http://scansits.mit..edu/motifscan....seg.html

RESULTS

The minimal critical region of PE on 10q22 consists of a 444 kb fragment located within a female recombination hotspot and is highly enriched for DNA/RNA binding proteins

17 genes in the critical region (11.7 Mb) with maternal effect located between CTNNA3-KCNMA1 were sequenced to identify nucleotide variations in affected sisters (n=16) with preeclampsia. Besides the CTNNA3 and KCNMAl genes, this included LRRTM3, JDPl, SIRTl, HERC4, MAWBP, HNRPH3, RUFY2, DNA2L, SLC25A16, CXXC6, CCARl, C10orf24, DDX50, SUPV3L1, and MYST4. Nucleotide variations were identified by sequencing known and predicted exons and 30-50 bases of flanking intron sequences amplified from genomic DNA of affected sister pairs from eight informative families with DNA available from 3 generations (affected sisters, their parents and the children born from preeclamptic pregnancies) . Fifty-five diallelic variations were found in affected sisters: 53 single nucleotide and 2 insertion/deletion polymorphisms (Fig. 1) . Using these for haplotype analysis, the minimal critical region of 10.q22, i.e., the region with maximal sharing of alleles between affected sisters of the same family, was found to reside in a 444 kb fragment bordered by RUFY2 and CCARl (Fig. 1) . Strikingly, all genes in this region except one (SLC25A16) code for DNA binding proteins (Fig. 2, columns A, B) . Moreover, two genes (DDX50, HNRPH3) flanking the minimal critical region also code for nucleotide binding proteins, yet with specificity for single stranded nucleotides, i.e. KNA. Thirdly, the minimal critical region resides within a female-specific recombination hotspot recently identified between D10S767and D10S599 following genome wide comparison of the genetic and physical maps of chromosome 10 (Fig. 1) . Chromosomal regions with sex specific differences in meiotic recombination frequencies are associated with imprinted chromosomal regions. (Paldi A, et al., Curr. Biol. 5: 1030-1035, 1995) .

Identification of a novel member of the winged helix family within, the minimal critical region.

Rather than correcting the minimal critical region for the maternal effect by scoring for maternally derived alleles only (requiring additional analysis of parental alleles) , it was preferred to identify preeclampsia candidate genes by expression screening. Out of nine genes, the expression of two, MAWBP and C10orf24, was downregulated in complete hydatid!form moles compared to normal placentas of identical gestational age (Fig. 2, columns C, D) . All nine genes are expressed in the early human placenta.

In MAWBP, heterozygous nucleotide variations leading to amino acid changes were not found in all families nor, when present, observed in both affected sisters from the same families (Fig. 1) . In contrast, in C10orf24, heterozygous mutations, both missense (n=4) and silent (n=3), were seen in all families and always identical between affected sisters of the same family (Fig. 1) . All four amino acid changes were predicted to be mutagenic (SIFT v.2 program) (Ng C, et al. , Genome Res. 12: 436-446, 2002) . One substitution (Y153H) was predominant, being found in 7 out of 8 families. The Y153H mutation resides in exon 2 within a protein domain with strong homology (27% identity, 59% similarity) to the C. elegans HAM-I protein previously demonstrated to be involved in abnormal migration.

A complete transcript analysis was performed by RT- PCR using an informative set of intron spanning primers complementary to known and predicted exons (NCBI, Ensemble, Nedo) covering 70,000 bp on 10q22.1. Out of six potential ClOorf 24 transcripts (Fig. 3a), four (GenBank Accession numbers: AY842014, AY842015, AY842016, AY842017) are transcribed in the early placenta including invasive extravillus trophoblast (Fig. 3b) . Exons 1 and 2 are separated by a large intron (54 kb) , but shared between all placental transcripts. Indeed, a single 1505 bp CpG island (74 % GC, CpG obs/exp ratio 0.92) is present covering nucleotides -521 to + 984 relative to the start codon in exon 1. The first four nucleotides (ATGG) of the coding region in exon 1 are followed by a 336 bp sequence, immediately followed by the same 336 bp sequence present as a perfect direct repeat. The first repeat is transcribed, spliced and translated. The second repeat retains the same ORF, but is not transcribed.

By variable donor splice site usage in exons 3 and 4, exon 3 skipping and the use of a second untranslated region encoded by exon 5, three different proteins (isoforms A, B and C) are generated (989, 227 and 169 amino acids, respectively) (Fig. 3a) . Conventional blast analysis of these proteins generated no hits with any known gene family nor with conserved domains, except for the presence of a paralogous gene (DKFZp762K222) on chromosome 4 in addition to homologous genes with unknown function in other species.

However, secondary structure analysis (Psipred) predicted a domain of 87 amino acids (encoded by exon 2 and part of exon 3) characteristic of a DNA binding winged helix domain (Helix 1-Sheetl-Helix 2-loop-Helix 3-Sheet 2-Wing 1-Sheet3-Wing 2) .

Alignment of this domain in the C10orf24 protein with all currently known members of the forkhead (FOX) class of winged helix genes showed a striking similarity in domain organisation (Table 3) . The presence and position of conserved hydrophobic amino acids that control wing 2 stability and as such influence DNA binding are conserved in both protein families. Nuclear localization (LARAASEL) and nuclear export (KFGFSLLWESLSRKEK) signals were predicted in exons 1 and 3, respectively. The NLS motif forms part of a bihelical structure.

In FOX proteins, these dual control sequences, besides other factors, regulate nuclear/cytoplasmic shuttling of FOX proteins. Nuclear exclusion targets FOX proteins for degradation by ubiquitination with inactivation of the phosphatidylinositol 3-kinase (PI3-K) and protein kinase B (PKB, also called Akt) pathway. Amino acids reactive with kinases of the PI3-K and PKB families were predicted in the C10orf24 protein by Motif Scan under high stringency search conditions (T516: CAMK2G; T141: ATM and PRKDC; S812: PRKDC and S764: CSNK1G2) .

Alignment of the winged helix domains showed that all FOX genes follow an absolutely conserved Y/F Y/F-6-W-7-L-5-F rule (Y/F Y/F-11-W-7-L-5-F in FOXO genes) (Table 3A), while this is not seen in C10orf24 (Table 3B) . Following transfection of extravillus trophoblast cells with GFP recombinant proteins, the two shortest isoforms (B and C) , that do not contain the NES, but retain the NLS sequence, are localized in the nucleus only (Fig. 3C) . Within the nucleus, isoform C lacking the second part of the winged helix domain is localized at discrete nuclear sites, i.e. the nucleoli. Isoform A encoded by the largest transcript (AY842014) is found in either the nucleus or in the cytoplasm (Fig. 3C) . This pattern is exclusive: no cells were seen with combined nuclear and cytoplasmic expression. Moreover, nuclear expression of isoform A is restricted to polyploid trophoblast cells (Fig. 3C) .

C10orf24 is a candidate gene for preeclampsia in the Netherlands

The C10orf24 gene fulfils multiple criteria for a gene responsible for preeclampsia in the Dutch population.

Given the maternal effect, causal relation of this gene with preeclampsia can only exist if the mutations, which, by imprinting, can be heterozygous in the preeclamptic mother, are maternally derived with transmission to the children (i.e. placenta) born from the affected pregnancies. If imprinting is the non-Mendelian mechanism involved, expression of one or more C10orf24 transcripts in the placenta is from the maternal allele only. Transmission screening of missense mutations in eight preeclamptic families with DNA available from 3 generations showed matrilineal transmission from mother to affected daughter pairs in all cases, where informative combinations of hetero- and homozygosity permitted discriminative tracing (Fig. 4) . Moreover, the single exception present confirmed this rule: in family PE9046 where only one affected sister carried the R18P mutation in exon 1, this mutation is derived from the father (Fig. 4) . Additional analysis of the Y153H mutation in 32 preeclamptic sisters included previously in a linkage study confirmed this transmission distortion towards the C allele (Oudejans C. et al. , MoI. Hum. Reprod.) .

Heterozygous CT and homozygous CC genotypes were present in a near 50-50 % ratio. Only two sisters (4%) from one family contained the TT wildtype in a total of 48 patients analyzed (Table 3, Fig. 4) . In controls (n=32), however, consisting of females of similar Kaukasian origin with no history of preeclampsia, and uncorrected for matrilineal inheritance, the Y153H variation was found also, • yet with normal Hardy Weinberg distributions of the genotypes (25-50-25 %) . The presence of CT heterozygotes in the normal population is compatible with a maternal effect gene subject to imprinting. The presence of homozygous CC genotypes in the normal population contradicts this model assuming that the C allele is fully penetrant. However, by conservation criteria, the predominant Y153H mutation is highly mutagenic. At its corresponding position, either a Y or F is found in all FOX proteins (Table 3A) and when changed mutagenic. Absolute conservation is also seen in the C10orf24 protein and its homologues (Table 3B) . Therefore, the presence of H at position 153 in the winged helix domain of the C10orf24 protein is non-conservative.

According to the present invention, a candidate gene, C10orf24, as well as marker nucleic acids derived therefrom, thus have been identified which can suitably be used in the diagnosis, monitoring, prevention and/or treatment of preeclampsia.

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Claims

1. Method for predicting and/or diagnosing a pregnancy at risk for preeclampsia and/or related disorders, comprising:

(a) obtaining at least one marker nucleic acid from a biological sample;

(b) providing the diagnosis based on at least one of the quantity, activity and/or sequence of said marker nucleic acid in said sample.

2. Method according to claim 1, wherein providing the diagnosis comprises:

- determining the sequence of at least one marker nucleic acid/ and

- detecting the presence or absence of at least one mutation and/or modification in said marker nucleic acid.

3. Method according to claim 1, wherein providing the diagnosis comprises comparing the quantity of at least one marker nucleic acid to the quantity of at least one reference nucleic acid.

4. Method according to claim 1, wherein providing the diagnosis comprises determining the activity of at least one marker nucleic acid.

5. Method according to any of the claims 1-4, wherein the marker nucleic acid is a placental marker nucleic acid.

6. Method according to any of the claims 1-5, wherein the marker nucleic acid is derived from a gene from the 10q22 region selected from the group consisting of MAWBP (Accession NO: NM_022129), HNRPH3 (Accession NO: NM_012207), RUFY2 (Accession NO: NM_017987) , DNA2L (Accession NO: XM_166103), SLC25A16 (Accession NO: NM_152707), CXXC6 (Accession NO: NM_030625), CCARl (Accession NO: NM_018237), C10orf24 (SEQ ID- No: 1), and DDX50 (Accession NO: NM_024045) , and/or fragments, allelic variants and/or the complements thereof.

7. Method according to claim 6, wherein the marker nucleic acid is derived from C10orf24.

8. Method according to claim 7, wherein the marker nucleic acid is derived from C10orf24.1, C10orf24.2, C10orf24.3, and/or C10orf24.4 (Accession Nos: AY842014, AY842015, AY842016, AY842017), and/or fragments, allelic variants and/or the complements thereof.

9. Method according to claim 3, wherein the reference nucleic acid is a placental reference nucleic acid.

10. Method according to claim 9, wherein the placental reference nucleic acid is derived from the gene GCMl (Accession NO: NM_003643) , or a fragment, allelic variant and/or the complements thereof.

11. Method according to any of the claims 1-10, wherein the biological sample is a sample of a maternal bodily fluid.

12. Method according to claim 11, wherein the sample of maternal bodily fluid is a blood sample obtained from a pregnant woman obtained at least prior to week 17, 16, 15, 14, 13, 12, 11, 10, 9 or 8 of gestation.

13. Marker nucleic acid for use in predicting, diagnosing, preventing and/or treating preeclampsia and/or related disorders, wherein the placental marker nucleic acid is derived from C10orf24 (Accession Nos : AY842014, AY842015, AY842016, AY842017), and/or fragments, allelic variants and/or the complements thereof.

14. Kit for performing a method according to any of the claiMs 1-12, comprising priMers and/or probes for the detection and/or amplification of one or more of the marker nucleic acids .

15. Kit as claimed in claim 14, wHerein the primers and/or probes hybridize to a marker nucleic acid derived from a gene on 10q22, selected from the group consisting of MAWBP (Accession NO: NM_022129) , HNRPH3 (Accession No: NM_012207), RUFY2 (Accession NO: NM_017987), DNA2L (Accession NO: XM_166103), SLC25A16 (Accession NO: NM_152707) , CXXC6 (Accession NO: NM_030625), CCARl (Accession NO: NM_018237), C10orf24 (Accession Nos: AY842014, AY842015, AY842016, AY842017), and DDX50 (Accession NO: NM_024045) , and/or fragments, allelic variants and/or the complements thereof.

16. Kit as claimed in claim 14 or 15, wherein the primers and/or probes hybridize to a placental marker nucleic acid derived from C10orf24.1, C10orf24.2, C10orf24.3 and/or C10orf24.4 (Accession Nos: AY842014, AY842015, AY842016, AY842017) .

17. Kit as claimed in claim 14, 15 or 16, wherein the primers comprise at least part of the sequences identified in Table 1 and 2.

18. Kit as claimed in claim 14-18, further comprising primers or probes used for the detection of at least one placental reference nucleic acids.

19- Kit as claimed in claim 18, wherein the placental reference nucleic acid is derived from GCMl (Accession NO: NM_003643) .

20. Primer for use in a method according to any of the claims 1-12, comprising at least part of the sequences identified in Table 1 and 2.