WO2007097741A1

WO2007097741A1 - Method of diagnosing intrauterine growth restriction

Info

Publication number: WO2007097741A1
Application number: PCT/US2006/005632
Authority: WO
Inventors: Benjamin Tycko; Harshwardhan M. Thaker
Original assignee: Columbia University in the City of New York
Current assignee: Columbia University in the City of New York
Priority date: 2006-02-17
Filing date: 2006-02-17
Publication date: 2007-08-30
Anticipated expiration: 2008-08-17

Abstract

Intrauterine growth restriction (IUGR) ranks high among the most common and costly obstetrical conditions. Hospital-based and registry studies have consistently shown that intrauterine growth restriction (IUGR) predisposes to mortality, postnatal morbidity, and later complications, including acute and chronic pulmonary disease, necrotizing enterocolitis, intraventricular hemorrhage, and retinopathy of prematurity. The present invention provides a method of identifying genes which contribute to intrauterine growth restriction (IUGR), a method of diagnosing IUGR in a subject, and kits for diagnosing IUGR in a subject.

Description

METHOD OF DIAGNOSING INTRAUTERINE GROWTH RESTRICTION

SPECIFICATION 1. FIELD OF THE INVENTION

The present invention relates to a method of diagnosing intrauterine growth restriction (IUGR) in a subject, kits for diagnosing IUGR in a subject, and a method of identifying additional genes which contribute to IUGR.

2. BACKGROUND OF THE INVENTION

2.1. Intrauterine Growth Restriction (IUGR)

Antenatal growth restriction ranks high among the most common and costly obstetrical conditions. Hospital-based and registry studies have consistently shown that intrauterine growth restriction (IUGR) predisposes to mortality, postnatal morbidity, and later complications, including acute and chronic pulmonary disease, necrotizing enterocolitis, intraventricular hemorrhage, and retinopathy of prematurity. It has long been appreciated that IUGR is not a single disorder, but instead has various causes. Suspected etiologies include genetic factors, notably chromosomal aberrations and confined placental mosaicism (Wilkins-Haug et al. 1997; Robinson et al. 1997; and Kalousek et al. 1994), and maternally transmitted infections such as cytomegalovirus, multiple gestations, underlying maternal medical conditions such as sickle cell anemia or lupus, and the effects of environmental toxins, notably maternal cigarette smoking (Bianci et al. 2000). But many cases of IUGR remain idiopathic, and maternal vascular under-perfusion (alternatively referred to as uteroplacental insufficiency) is an important factor in these cases. 2.2. Genetic Imprinting

Imprinting is an epigenetic phenomenon that differentially marks the chromatin in male versus female gametes by cytosine methylation and/or histone modifications, leading to monoallelic expression of certain genes in the offspring. Genes found in the gametes are "marked" via the cytosine methylation and/or histone modifications, and are inactivated in the new embryo. The genetic modification is believed to prevent binding of transcription factors to the promoter of the gene, thereby inactivating expression. Imprinted genes in embryos will have active maternal alleles and inactive paternal alleles, or vice versa. In contrast, most genes, which are not imprinted, will have active maternal alleles and active paternal alleles.

Examples of genetic imprinting include insulin-like growth factor 2 (IGF2), which is imprinted on the maternal side and nonimprinted on the paternal side. The IGF2 receptor (IGF2r) is imprinted on the paternal side and nonimprinted on the maternal side. Thus, the paternal IGF2 allele is expressed and the maternal IGF2 allele is repressed, whereas the maternal IGF2r allele is expressed and the paternal IGF2r allele is repressed. Failure of genetic imprinting has been implicated in cancer and some congenital disorders.

2.3. Imprinted Genes And Non-imprinted Genes As Targets For IUGR

Diagnostics Genes subject to parental imprinting are interesting candidates for a role in IUGR. Data from mutant mice and rare human syndromes indicate that imprinted genes often control growth, and a survey of such genes indicates a strong correlation between the direction of imprinting (i.e., the parental origin of the expressed allele) and the effect on growth, with paternally expressed/maternally repressed imprinted genes promoting growth and maternally expressed/paternally repressed imprinted genes retarding growth (Tycko et al. 2002). Imprinted genes on distal mouse chromosome 7 (corresponding to human chromosome 1 Ip 15.5), including Igf2, Cdknlc and Phlda2 all control placental and fetal growth in mice, as proven by knockout and transgenic experiments (Caspary et al. 1999; Takahashi et al. 2000; Kanayama et al. 2002; Eggenschwiler et al. 1997; Frank et al. 2002; Salas et al. 2004), and such quantitative effects on placental and fetal growth are also observed for imprinted genes on other chromosomes, including the MEST gene on human chromosome 7q32 and mouse chromosome 6 (Tycko et al. 2002). IUGR in humans can result from rare uniparental chromosomal disomies (UPDs) for chromosomes containing imprinted genes, wherein the fetus inherits two copies of a chromosome pair from one parent, notably maternal UPD7 in the Silver-Russell syndrome (Monk et al. 2002).

Notwithstanding the foregoing, it has been unclear whether imprinted genes are implicated in non-syndromic human IUGR.. One study, using radioimmunoassay of fetal blood, found a reduction in mean insulin-like growth factor-2 levels in fetuses that were small for gestational age, but failed to find a significant reduction in fetuses that were classified as IUGR based on poor placental perfusion ascertained by Doppler ultrasound (Holmes et a 1997). Aside from IGF2, other imprinted genes have not been intensively studied in non-syndromic IUGR.

2.4. Current IUGR Diagnostic Approaches

IUGR is typically defined as a fetus or newborn infant whose weight is below the 10th percentile for its gestational age. Currently, the primary test used for diagnosing IUGR is ultrasound exam, where growth of the fetus is measured and the amount of amniotic fluid is estimated. Determining the growth of the fetus during an ultrasound exam is done by measurement of the fetus' head, abdomen, and legs. Small size of the fetus and low amounts of amniotic fluid are indicative of IUGR. Certain behaviors by the mother and other factors will increase the likelihood that a fetus or newborn infant will have IUGR. The behaviors and risk factors which can increase the likelihood of IUGR include: poor nutrition, heart disease, high blood pressure, smoking, drug abuse, and alcohol abuse. Congenital or chromosomal abnormalities and infections during pregnancy may also contribute to poor fetal growth. However, all small babies do not necessarily have IUGR; some babies are simply constitutionally small, but are otherwise normal. It is often difficult for doctors to differentiate between babies which have IUGR and those which are constitutionally small. In some cases, termed asymmetric IUGR, babies with IUGR exhibit asymmetric growth, and are anatomically disproportionate when compared to healthy babies. Babies with asymmetric IUGR may also have underdeveloped organs and tissues. In other cases, termed symmetric IUGR, babies have normally proportioned anatomies, but are small for their gestational age. Other factors, such as the level of amniotic fluid, must be considered in order to conclude that a baby suffers from IUGR. In many cases, concluding that a baby is constitutionally small may only be done once a diagnosis of IUGR has been excluded.

Because the definition and method of diagnosis for IUGR is subjective in nature, and requires estimation of weight and comparison with other newborns of similar gestational age, diagnosing IUGR requires close monitoring of fetal growth and development. Furthermore, accurate dating of the gestational age is needed for a reliable diagnosis of IUGR. Due to these subjective factors, a diagnosis of IUGR is often made only after the baby has been born. Because IUGR can result in complications during childbirth, and sometimes may lead to death, it is imperative that IUGR is diagnosed as early as possible. Thus, there is a need for a method for accurately and objectively diagnosing IUGR during or after pregnancy. It has been found that a discrete subset of various imprinted and non-imprinted genes in late- gestation placental tissue from non-syndromic human IUGR manifest altered expression. Comprehensive mRNA profiling analysis has identified certain imprinted genes, as well as a specific subset of non-imprinted genes, as dysregulated in IUGR. Accordingly, the present application provides for a method of diagnosing IUGR via measurement of expression of these IUGR-related genes in placental tissue, and the protein products of a subgroup of these genes, which are secreted from the placenta into the circulating maternal blood. The present application also provides for a method of identifying genes related to IUGR. The present application also provides kits for the diagnosis of IUGR in a subject.

3. SUMMARY OF THE INVENTION

The present invention relates to a method of diagnosing IUGR in a subject by measurement of expression of intrauterine growth restriction (IUGR)- related genes, kits for use in diagnosing IUGR in a subject, and a method of identifying additional genes relating to IUGR.

The present invention is based in part on the discovery that various genes are upregulated or downregulated in subjects diagnosed with IUGR. These genes are referred to herein as IUGR-related or IUGR-associated genes. In particular, it has been discovered that determination of elevated or reduced expression levels of IUGR-related genes in maternal or fetal samples (e.g., tissue or fluid samples) can be a reliable predictor of IUGR. As such, the methods of the present invention are useful as an invaluable diagnostic tool for identifying babies at risk for IUGR, which would allow for treatment of the babies at risk for IUGR prior to birth. The ability to intervene prior to birth can provide numerous health benefits to both the child and the mother, and may reduce health care costs due to treatment of health complications associated with IUGR.

Diagnostic assays may be performed utilizing tissue or fluid samples obtained from the fetus or the mother. Fetal gene expression can be determined from fetal tissue or fluid samples by isolating nucleic acids (i.e., DNA or RNA) or proteins from the fetal tissue or fluid samples. Fetal gene expression from maternal tissue or fluid samples may be determined by isolating fetal nucleic acids or fetal proteins found in maternal tissue or fluid samples. Maternal tissue or fluid samples include, but are not limited to, placenta, blood, and plasma samples. Maternal plasma is known to contain fetal nucleic acids and fetal cells, which may be isolated and assayed according to the present invention to diagnose the fetus for IUGR. (Poon et al. , van Wijk et ah). Diagnosis of IUGR in a fetus may be performed in a clinical or laboratory setting. The tissue and/or fluid samples may be extracted, for example, by a physician or a laboratory technician. Analysis of gene expression in the tissue and/or fluid samples may be performed immediately, or alternatively the tissue and/or fluid sample may be preserved for later analysis. Gene expression may then be compared against control samples or against a database containing normative gene expression values for particular genes.

The diagnostic test may target one or more IUGR-related gene(s). The present invention identifies several IUGR-related genes which may be targeted in the diagnostic test. Utilizing the methods of the present invention, additional IUGR- related genes may be identified for use in diagnosis of IUGR. Maternally or paternally imprinted genes are candidates for analysis, as they are often to be associated with promotion or restriction of fetal growth. The IUGR-related genes targeted by the diagnostic test include the genes disclosed in the present invention, or may be genes identified via the methods of the present invention.

The present invention also provides for a method of diagnosing intrauterine growth restriction comprising (a) measuring the level of gene expression of an intrauterine growth restriction related gene in a subject sample; (b) comparing the level of gene expression of the target gene in the subject sample with a normalized gene expression level representing the gene expression level of the target gene derived from one or more healthy subject(s) without intrauterine growth restriction; wherein a statistically significant decrease or increase in the level of gene expression of the target gene in the subject sample with intrauterine growth restriction when compared to the normalized gene expression level of the target gene derived from healthy subjects without intrauterine growth restriction indicates a diagnosis of intrauterine growth restriction. The level of gene expression in one or more healthy subject(s) may be measured in a side-by-side comparison or can be a previously determined value, for example, obtained using a plurality of healthy subjects.

The present invention also provides for a method of diagnosing intrauterine growth restriction comprising (a) measuring the level of gene expression of a maternally expressed/paternally repressed imprinted gene and the level of expression of a maternally repressed/paternally expressed imprinted gene in a subject sample; (b) calculating the ratio of gene expression of the maternally expressed/paternally repressed imprinted gene to the maternally repressed/paternally expressed imprinted gene in the subject sample; (c) comparing the ratio of gene expression of the target maternally expressed/paternally repressed imprinted gene to the target maternally repressed/paternally expressed imprinted gene with a normalized ratio of gene expression of the target maternally expressed/paternally repressed imprinted gene to the target maternally repressed/paternally expressed imprinted gene in one or more healthy subject(s) without intrauterine growth restriction; wherein a statistically significant decrease or increase of the ratio of gene expression of the target maternally expressed/paternally repressed imprinted gene to the target maternally repressed/paternally expressed imprinted from the subject sample when compared to the ratio of gene expression from the normalized ratio of gene expression derived from a healthy subject sample indicates a diagnosis of intrauterine growth restriction. The ratio of gene expression in one or more healthy subject(s) may be measured in a side-by-side comparison or can be a previously determined value, for example, obtained using a plurality of healthy subjects.

The present invention also provides for a method of diagnosing IUGR in a subject by measuring gene expression in a tissue or fluid sample derived from a mother, a fetus, or a newborn infant. The tissue or fluid sample may be placental tissue, amniotic tissue, or blood. As defined herein, "tissue" encompasses individual cells.

The present invention provides for a method of diagnosing IUGR in a subject by measuring the gene expression of an IUGR-associated gene selected from the group consisting of CD81, CDKNlC, DCN, DIO3, DLKl, GATM, GNAS, GRBlO, HYMAI, IGF2, IGF2R, MEG3, MEST, MKRNl, NDN, NNAT, MESTO, PEG3, PLAGLl, PON2, PPPlCC, SGCE, SNRPN, SNURF, PHLDA2, UBE3A, ZIM2, AGTRl, CRH , DSCRl, GLRX, HPGD, IGFl₅ INDO, INHBA, LEP, PSG4 or a functionally equivalent gene. The present invention also provides for a method of diagnosing IUGR in a subject by measuring the gene expression of an IUGR- associated gene identified in Tables 2 or 3, or a functionally equivalent gene. In a preferred embodiment, the IUGR related gene measured is PHLD A2 and/or MEST, or functionally equivalent genes. In another preferred embodiment, the ratio of PHLD A2 to MEST is calculated.

The present invention also provides for methods of diagnosing IUGR in persons at risk of IUGR, including but not limited to, persons exhibiting one or more of the following conditions: poor nutrition, heart disease, high blood pressure, smoking, drug abuse, alcohol abuse, viral or bacterial infections, congenital or chromosomal abnormalities, maternal medical conditions such as sickle cell anemia or lupus, multiple gestations, and exposure to environmental toxins. The presence of one or more of the aforementioned risk factors can increase the likelihood of IUGR in the fetus or newborn infant, and makes mothers exhibiting said conditions suitable targets for the method of diagnosis of the present invention. Persons of ordinary skill in the art will recognize that many factors contribute to IUGR, and that other risk factors are well known and may be considered when performing the method of the present invention.

The present invention provides a method of identifying genes relating to intrauterine growth restriction comprising: (a) measuring the level of gene expression of a target gene in a subject sample diagnosed with intrauterine growth restriction; (b) measuring the level of gene expression of the target gene in a healthy subject sample not suffering from intrauterine growth restriction; (c) comparing the gene expression of the target gene in the subject sample with intrauterine growth restriction to the gene expression of the target gene in the healthy subject sample; wherein a statistically significant decrease or increase in gene expression of the target gene in the subject sample with intrauterine growth restriction when compared to the gene expression of the target gene in the healthy subject sample indicates a gene relating to intrauterine growth restriction. The present invention also provides for a method of identifying genes relating to IUGR by measuring gene expression in a tissue or fluid sample derived from a mother, a fetus, or a newborn infant. The tissue or fluid sample may be placental tissue, amniotic tissue, or blood. The present invention also provides for kits for the diagnosis of IUGR, comprising (1) oligonucleotide probes directed to intrauterine growth restriction related genes; (2) reagents and equipment for measuring gene expression; and (3) control reagents.

DEFINITIONS

As used herein, the term "cDNA" can refer to a single-stranded or double-stranded DNA molecule. For a single-stranded cDNA molecule, the DNA strand is complementary to the messenger RNA ("mRNA") transcribed from a gene. For a double-stranded cDNA molecule, one DNA strand is complementary to the mRNA and the other is complementary to the first DNA strand.

As used herein, a "coding sequence" or a "nucleotide sequence encoding" a particular protein is a nucleic acid molecule which is transcribed and translated into a polypeptide in vivo or in vitro when placed under the control of appropriate regulatory sequences. The regulatory sequences may include a control sequence which "directs the transcription" of the coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence. A coding sequence can include, but is not limited to, prokaryotic nucleic acid molecules, cDNA from eukaryotic mRNA, genomic DNA from eukaryotic {e.g. mammalian) sources, viral RNA or DNA, and even synthetic nucleotide molecules.

As used herein, the term "control sequences" refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers and the like, and untranslated regions (UTRs) including 5'-UTRs and 3'-UTRs, which collectively provide for the transcription and translation of a coding sequence in a host cell. As used herein, the term "gene" refers to a DNA molecule that either directly or indirectly encodes a nucleic acid or protein product that has a defined biological activity. Such genes may also be referred to as "biologically active" genes.

As used herein, the term "genomic DNA" refers to a DNA molecule from which an RNA molecule is transcribed. The RNA molecule is most often a messenger RNA (mRNA) molecule, which is ultimately translated into a protein that has a defined biological activity, but alternatively may be a transfer RNA (tRNA) or a ribosomal RNA (rRNA) molecule, which are mediators of the process of protein synthesis. As used herein, two nucleic acid molecules are "functionally equivalent" when they share two or more quantifiable biological functions. For example, nucleic acid molecules of different primary sequence may encode identical polypeptides; such molecules, while distinct, are functionally equivalent. In this example, these molecules will also share a high degree of sequence homology. Similarly, nucleic acid molecules of different primary sequence may share activity as a promoter of RNA transcription, wherein said RNA transcription occurs in a specific subpopulation of cells, and responds to a unique group of regulatory substances; such nucleic acid molecules are also functionally equivalent.

As used herein, two nucleic acid molecules are "homologous" when at least about 60% to 75%, 80%, 90%, or 95% of the nucleotides comprising the nucleic acid molecule are identical over a defined length of the molecule, as determined using standard sequence analysis software such as Vector NTI, GCG, or BLAST. DNA sequences that are homologous may be identified by hybridization under stringent conditions, as defined for the particular system. Defining appropriate hybridization conditions is within the skill of the art. See e.g. Current Protocols in Molecular

Biology, Volume I₅ Ausubel et ah, eds. John Wiley:New York NY, first published in 1989 but with annual updating, wherein maximum hybridization specificity for DNA samples immobilized on nitrocellulose filters may be achieved through the use of repeated washings in a solution comprising 0.1-2 x SSC (15-30 mM NaCl, 1.5-3 niM sodium citrate, pH 7.0) and 0.1 % SDS (sodium dodecylsulfate) at temperatures of 65- 68°C or greater. For DNA samples immobilized on nylon filters, a stringent hybridization washing solution may be comprised of 40 mM NaPO₄, pH 7.2, 1-2% SDS and 1 mM EDTA. Again, a washing temperature of at least 65-68⁰C is recommended, but the optimal temperature required for a truly stringent wash will depend on the length of the nucleic acid probe, its GC content, the concentration of monovalent cations and the percentage of formamide, if any, that was contained in the hybridization solution (Ausubel et ah, supra). As used herein, the term "nucleic acid molecule" includes both DNA and RNA and, unless otherwise specified, includes both double-stranded and single- stranded nucleic acids. Also included are molecules comprising both DNA and RNA, either DNA/RNA heteroduplexes, also known as DNA/RNA hybrids, or chimeric molecules containing both DNA and RNA in the same strand. Nucleic acid molecules of the invention may contain modified bases. The present invention provides for nucleic acid molecules in both the "sense" orientation (i.e. in the same orientation as the coding strand of the gene) and in the "antisense" orientation (i.e. in an orientation complementary to the coding strand of the gene).

As used herein, the term "sequence" refers to a nucleic acid molecule having a particular arrangement of nucleotides, or a particular function, e.g. a termination sequence.

As used herein, the term "subject" refers to an animal, e.g. , a mammal. In one embodiment, the subject is a human. In another embodiment, the subject is a human fetus or a newborn infant. In another embodiment, the subject is a pregnant human female.

As used herein, the term "derived" means "obtained from," "descending from," or "produced by." In the context of tissue or fluid samples derived from a particular parent source, the term derived refers to obtaining the tissue or fluid samples from the parent source. In the context of nucleic acids or polypeptides derived from a particular parent source, the term derived refers to the use of the parent source as a template for the nucleic acid sequence or the amino acid sequence. The nucleic acid or polypeptide derived from the parent source may possess all or part of the nucleic acid or amino acid sequence of the parent source, in the presence or absence of deletions, substitutions, or modification. In the context of a gene expression value of a target gene derived from subjects, the term derived refers to the sampling of multiple subjects to obtain a normalized, average value.

As used herein, the term "probe" refers to a nucleic acid oligomer that hybridizes specifically to a nucleic acid target sequence, under conditions that promote hybridization, thereby allowing detection of the target sequence. Detection may either be direct (i.e., resulting from a probe hybridizing directly to the target sequence) or indirect (i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe and target sequences). The "target sequence" of a probe refers to a sequence within a nucleic acid, preferably in an amplified nucleic acid, which hybridizes specifically to at least a portion of a probe oligomer. A probe may hybridize under appropriate hybridization conditions even if not completely complementary to the target sequence, if the probe is sufficiently homologous to the target sequence. The probe may be labeled, i. e. , joined directly or indirectly to a detectable molecular moiety or a compound that leads to a detectable signal. Direct labeling can occur through bonds or interactions that link the label to the probe, including covalent bonds and non-covalent interactions (e.g. hydrogen bonding, hydrophobic and ionic interactions), or formation of chelates or coordination complexes. Indirect labeling occurs through use of a bridging moiety (a "linker"), that joins a label to the probe, and which can amplify a detectable signal (e.g., see PCT No. WO 95/16055 (Urdea et al.)). Labels are well known and include, for example, radionuclides, ligands (e.g., biotin, avidin), enzymes and/or enzyme substrates, reactive groups, redox active moieties such as transition metals (e.g., Ruthenium), chromophores (e.g., a moiety that imparts a detectable color), luminescent compounds (e.g., bioluminescent, phosphorescent or chemiluminescent labels) and fluorescent compounds. Those skilled in the art will appreciate that a labeled probe may be a mixture of labeled and unlabeled oligonucleotides that hybridize specifically to the target sequence, to optimize the specific activity of the probe reagent for detection.

4. BRIEF DESCRIPTION QF THE FIGURES

Figure 1. Northern blot analysis of PHLD A2 and MEST mRNA in IUGR and non-IUGR placentae. Examples of cases of IUGR with unbalanced expression of PHLD A2 relative to MEST are shown in the left panel. See Figure 2 for the complete Northern blotting data. The right panel shows consistency of the PHLDA2/MEST mRNA ratio across three cotyledons of a single normal term placenta. Figure 2. The PHLDA2/MEST mRNA expression ratio in IUGR and non-IUGR placentae. A, The PHLDA2/MEST mRNA ratio was determined by Phosphorimaging of Northern blots and plotted as a function of gestational age. Cases of IUGR associated with developmental anomalies or with twin pregnancies are indicated. B₅ PHLDA2/MEST plotted as a function of placental weight. Placental weights are reduced overall in the cases with IUGR₅ but the PHLDA2/MEST mRNA ratio is elevated in IUGR compared to non-IUGR independently of placental weight.

Figure 3. Analysis of KvDMRl DNA methylation by Southern blotting. Genomic DNAs from IUGR and non-IUGR placentae were digested with the non-methylation-sensitive restriction enzyme Pstl (P) either with or without the methylation-sensitive restriction enzyme Smal (S). Bands corresponding to the imprinted (methylated) and non-imprinted (unmethylated) alleles are indicated (see Dao et al. 1999 for the restriction map of this region). The placenta from BWS is a positive control for abnormal KvDMRl methylation; this case shows a substantial loss of the upper (methylated) band, while the IUGR and non-IUGR placentae show a normal pattern of equal upper and lower band intensities.

Figure 4. Immunoperoxidase staining for PHLD A2 protein in age- matched non-IUGR and IUGR placentae. A, Non-IUGR placenta at 28 weeks gestation. B, IUGR placenta at 28 weeks gestation. The arrows indicate PHLD A2- positive villous cytotrophoblast. The IUGR placenta shows abundant syncytiotrophoblast knots (arrowhead).

Figure 5. Validation of the differential expression of MEG3 RNA in IUGR vs. non-IUGR placenta by Northern blotting. A, Example of Northern blotting showing variation in MEG3 RNA expression, with low expression in an IUGR placenta. The blot was re-hybridized with a GAPDH probe for normalization. B, MEG3 RNA expression in 30 IUGR and 46 non-IUGR placentae measured by Phosphorimaging of the Northern blots, normalized to GAPDH mRNA. The physiological increase in placental MEG3 RNA that occurs near term in non-IUGR is absent or reduced in IUGR. The mean value of MEG3 mRNA in IUGR placentae in this Northern blotting series was 0.17 and the mean value in non-IUGR placentae was 0.41. A univariate analysis of variance adjusting for gestational age showed that this difference was significant at p = 0.015. Figure 6. Dendrogram of genes differentially expressed in IUGR with uteroplacental insufficiency. Genes are displayed on the Y-axis and cases (IUGR and non-IUGR placentae) on the X-axis. The color scale corresponds to normalized mRNA expression from 0 to 4, relative to the experiment mean, with red representing high, yellow intermediate and blue low expression. All genes passing the ANOVA test with a Benjamini-Hochberg false discovery rate of 0.05 are shown, and several genes discussed in the text are indicated.

5. DETAILED DESCRIPTION OF THE INVENTION

For the the purpose of clarity, and not by way of limitation, the detailed description of the invention is divided into the following subsections: (i) Methods of diagnosing IUGR in a subject;

(ii) Methods of identifying genes relating to IUGR; and

(iii) Kits for diagnosing IUGR.

The present invention is based, in part, on the identification of genes which are differentially expressed in fetuses diagnosed with IUGR or mothers carrying fetuses diagnosed with IUGR, when compared to normal, healthy fetuses or mothers bearing normal, healthy fetuses. The present invention relates to a method of diagnosing IUGR in fetuses and newborn infants, a method for identifying genes relating to IUGR, and kits used for the diagnosis of IUGR in fetuses and newborn infants. The present invention is useful for determining whether a fetus or newborn infant suffers from IUGR, allowing physicians to administer the appropriate treatment to the fetus or newborn infant and the mother. The present invention is also useful for the identification of genes which may allow for the diagnosis, prevention, or treatment of IUGR. The kits of the present invention are useful for the diagnosis of IUGR. 5.1. Methods of Diagnosing Intrauterine Growth Restriction in a Subject

The present invention provides for a method of diagnosing intrauterine growth restriction comprising: (a) measuring the level of gene expression of a intrauterine growth restriction related gene in a subject sample; (b) measuring the level of gene expression of the growth restriction related gene in one or more healthy subject sample(s) not suffering from intrauterine growth restriction; (c) comparing the level of gene expression of the growth restriction related gene in the subject sample with intrauterine growth restriction to the level of gene expression of the target gene in the healthy subject sample; wherein a statistically significant decrease or increase in gene expression of the target gene in the subject sample with intrauterine growth restriction when compared to the gene expression of the target gene in the healthy subject sample indicates a gene relating to intrauterine growth restriction. The level of gene expression in one or more healthy subject(s) may be measured in a side-by- side comparison or can be a previously determined value, for example, obtained using a plurality of healthy subjects. The level of gene expression in one or more healthy subject(s) may be normalized. Determination of expression ratios which may be indicative of IUGR is described above.

In another embodiment, the methods may comprise: A method of diagnosing intrauterine growth restriction comprising (a) measuring the level of gene expression of a maternally expressed/paternally repressed imprinted gene and the level of expression of a maternally repressed/paternally expressed imprinted gene in a subject sample; (b) calculating the ratio of gene expression of the maternally expressed/paternally repressed imprinted gene to the maternally repressed/paternally expressed imprinted gene in the subject sample; (c) measuring the level of gene expression of a maternally expressed/paternally repressed imprinted gene and the level of expression of a maternally repressed/paternally expressed imprinted gene in a healthy subject sample not suffering from intrauterine growth restriction; (d) calculating the ratio of gene expression of the maternally expressed/paternally repressed imprinted gene to the maternally repressed/paternally expressed imprinted gene in the healthy subject sample; and (e) comparing the ratio from the subject sample to the ratio from the healthy subject sample; wherein a statistically significant decrease or increase of the ratio from the subject sample when compared to the ratio from the healthy subject sample indicates a diagnosis of intrauterine growth restriction. Ratios may be determined by measuring the expression of the paternally expressed/maternally repressed gene, measuring the expression of the paternally repressed/maternally expressed gene, and dividing the expression level of the paternally expressed and maternally repressed gene with the expression level of the paternally repressed and maternally expressed gene. The ratio of gene expression in one or more healthy subject(s) may be measured in a side-by-side comparison or can be a previously determined value, for example, obtained using a plurality of healthy subjects. The ratio of gene expression in one or more healthy subject(s) may be normalized. Genes which are suitable targets for this method are those which are oppositely imprinted, i.e., where one gene is maternally imprinted/paternally non- imprinted, and the second gene is maternally non-imprinted/paternally imprinted. Preferably, the two genes selected are known to have opposite effects on placental growth, i.e. , where one gene promotes placental growth and the second gene restrains growth. Imprinted genes are discussed in more detail below.

In another embodiment, the methods may comprise: A method of diagnosing intrauterine growth restriction comprising (a) measuring the level of gene expression of an intrauterine growth restriction related gene in a subject sample; (b) comparing the gene expression of the target gene in the subject sample with a normalized gene expression value of the target gene derived from healthy subjects without intrauterine growth restriction; wherein a statistically significant decrease or increase in gene expression of the target gene in the subject sample with intrauterine growth restriction when compared to the normalized gene expression value of the target gene derived from healthy subjects without intrauterine growth restriction indicates a diagnosis of intrauterine growth restriction. Normalized gene expression values for target genes may be derived from healthy subject without intrauterine growth restriction prior to measuring gene expression in the subject sample. As used herein, the term "normalized gene expression value" or a "normative gene expression value" refers to a value derived from multiple samples from a given group, such that the mean value is reliably representative of the gene expression of the target gene in that group. Normalized gene expression values may vary from group to group, depending on factors such as age or sex. It will be within the abilities of a person of ordinary skill in the art to determine a normative gene expression value based upon a set of gene expression data comprising multiple samples.

Use of tissue and fluid samples and extraction of nucleic acids from the tissue and fluid samples may be performed as described below. Tissue and fluid samples may be obtained directly from the fetus to determine gene expression levels. Fetal tissue and fluid samples may also be obtained from maternal sources. Fetal DNA and/or RNA may be isolated directly from maternal plasma utilizing well- known techniques, such as RT-PCR. (See Poon et al. and Costa et ah). Alternatively, fetal cells may be isolated from maternal plasma, and fetal DNA, RNA, and/or proteins may be isolated from the fetal cells. (See van Wijk et al). Gene expression may be determined by any means known in the art, including the methods described in more detail below.

In one non-limiting embodiment, a maternal blood sample is drawn, and the plasma is separated utilizing standard techniques. Fetal nucleic acids, such as RNA, may be isolated from the maternal plasma. The fetal nucleic acids may be cell- free, or may be derived from fetal cells isolated from maternal sources. The fetal nucleic acids may then be assayed to determine gene expression of one or more IUGR-related gene(s). Any assay for gene expression known in the art may be utilized, including, but not limited to, RT-PCR or gene microarrays. Alternatively, fetal proteins isolated from fetal cells may be assayed to determine gene expression. Protein expression can be determined via any method known in the art, for example, by ELISA. Levels of gene expression may then be compared against a control sample, or may be compared against a database containing normative expression values for the targeted genes. Determining expression of IUGR-related genes may be performed as described below. In one non-limiting embodiment, gene expression may be determined utilizing real time polymerase chain reaction (RT-PCR). In another embodiment, determination of expression of IUGR-related genes is performed by Northern blot. In another embodiment, a GENECHIP™ may be used to determine expression. In a preferred embodiment, the method is performed to detect gene expression of one or more of the gene sequences identified in Table 2 and Table 3. In another preferred embodiment, gene expression is determined for one or more of the following genes: CD81, CDKNlC, DCN, DIO3, DLKl, GATM, GNAS₅ GRBlO, HYMAI, IGF2, IGF2R, MEG3, MEST, MKRNl₅ NDN, NNAT₅ MESTO₅ PEG3, PLAGLl, PON2, PPPlCC, SGCE₅ SNRPN₅ SNURF, PHLDA2, UBE3A, ZIM2, AGTRl, CRH , DSCRl, GLRX, HPGD, IGFl, INDO₅ INHBA, LEP, PSG4 or a functionally equivalent gene. Expression may also be determined from the IUGR- associated genes identified in Tables 2 or 3, or a functionally equivalent gene. In a preferred embodiment, gene expression of PHLD A2 and MEST genes, or functionally equivalent genes, is determined. Other methods of detecting gene expression include, but are not limited to, northern blots, phosphorimaging, southern blots, and dot blots. Genes which are suitable targets for diagnostic examination include genes which are differentially expressed in fetuses or newborn infants diagnosed with IUGR or mothers carrying fetuses diagnosed with IUGR, when compared to normal, healthy fetuses or newborn infants or mothers bearing normal, healthy fetuses. Differential expression of genes is discussed above. Non-limiting examples of target genes for diagnosis of IUGR in a subject may be found in Table 2 and Table 3. In another nonlimiting embodiment, the method of diagnosing intrauterine growth restriction is practiced in subjects at risk of IUGR. Persons at risk of IUGR include women exhibiting one or more of the following conditions: poor nutrition, heart disease, high blood pressure, smoking, drug abuse, alcohol abuse, viral or bacterial infections, congenital or chromosomal abnormalities, maternal medical conditions such as sickle cell anemia or lupus, multiple gestations, and exposure to environmental toxins.

5.2. Methods of Identifying Genes Relating To IUGR

5.2.1. Genetic Screening Techniques

The present invention relates to methods of identifying genes relating to IUGR. These methods may comprise: (a) measuring the level of gene expression of a target gene in a subject sample diagnosed with intrauterine growth restriction; (b) measuring the level of gene expression of the target gene in a healthy subject sample not suffering from intrauterine growth restriction; (c) comparing the gene expression of the target gene in the subject sample with intrauterine growth restriction to the gene expression of the target gene in the healthy subject sample; wherein a statistically significant decrease or increase in gene expression of the target gene in the subject sample with intrauterine growth restriction when compared to the gene expression of the target gene in the healthy subject sample indicates a gene relating to intrauterine growth restriction. Methods of performing statistical analysis are discussed in greater detail below. Gene expression may be determined by (a) obtaining a tissue or fluid sample from a subject diagnosed with intrauterine growth restriction; (b) extracting the RNA from the tissue or fluid sample; (c) determining the expression of genes via methods well known in the art.

Any tissue and fluid sample from a subject may be used. Tissue and fluid samples may be derived from the mother, the fetus, or the newborn infant. Examples of tissue samples that may be used include, but are not limited to: placenta, blood, plasma, and amniotic fluid. Tissue or fluid samples which may be useful for assaying the expression level of genes of interest are any tissues or fluids which may exhibit differential gene expression of the target genes. Preferred tissue or fluid samples include placental tissue, amniotic fluid, and blood. Tissue and fluid samples may be acquired via any method known in the art, including, but not limited to surgical excision, aspiration or biopsy. The tissue and fluid samples may be fresh or frozen.

Methods of purifying nucleic acids are well known in the art. See Sambrook et ah, Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989) at §§ 1.23-1.40, 2.73-2.80, 4.26-4.32 and 7.3-7.35). In a preferred embodiment, the nucleic acid extracted is RNA. In a preferred embodiment, RNA may be extracted using TRIZOL™ (Invitrogen, Carlsbad, California). As used in this application, the term "purifying" refers to separation of the target nucleic acid from one or more components of the biological sample (e.g., other nucleic acids, proteins, carbohydrates or lipids).

Preferably, a purifying step removes at least about 50%, more preferably about 70%, and even more preferably about 90% or more of the other sample components.

Nucleic acids isolated from the tissue or fluid samples may be amplified prior to the detection step. Methods of amplifying nucleic acids are well known in the art and have been described previously. For example, polymerase chain reaction (PCR) may be used to produce multiple copies of a target sequence. See U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159 (Mullis et al). RNA polymerase may also be used to amplify the target sequence. See U.S. Pat. Nos. 5,399,491 and 5,554,516 (Kacian et al), U.S. Pat. No. 5,437,990 (Burg et al), PCT Nos. WO 8801302 and WO 8810315 (Gingeras et al), U.S. Pat. No. 5,130,238 (Malek et al); and U.S. Pat. Nos. 4,868,105 and 5,124,246 (Urdea et al). Other methods include, but are not limited to, ligase chain reaction (LCR) and strand displacement amplification (SDA). See EP 0 320 308 and U.S. Pat. No. 5,422,252 (Walker et al). Determining expression of genes may be performed by use of methods known to those of ordinary skill in the art. Detection of nucleic acids isolated from the tissue or fluid samples may be used to determine expression of genes. Detection of nucleic acid sequences may be performed by any method known in the art. In a preferred embodiment, nucleic acids may be detected by hybridization with a complementary sequence, such as an oligonucleotide probe. See U.S. Pat. No. 5,503,980 (Cantor), U.S. Pat. No. 5,202,231 (Drmanac et al), U.S. Pat. No. 5,149,625 (Church et al), U.S. Pat. No. 5,112,736 (Caldwell et al), U.S. Pat. No. 5,068,176 (Vijg et al), and U.S. Pat. No. 5,002,867 (Macevicz)). Methods of detecting gene expression via hybridization with oligonucleotide probes include northern blots, phosphorimaging, southern blots, and dot blots. See Sambrook, supra. In a non- limiting example, detection via Northern blot may be used, and increased expression may be indicated by greater band intensity. Alternatively, an array of oligonucleotide probes assembled on a chip, referred to as a DNA chip, may be used to detect nucleic acids by hybridization. See U.S. Pat. Nos. 5,837,832 and 5,861 ,242 (Chee et al). An example of a DNA chip is the GENECHIP™, available from Affymetrix (Santa Clara, California). The DNA chip may contain oligonucleotide probes which are homologous to known genetic sequences, and are used to identify specific genes. Nucleic acids isolated from the tissue and fluid samples will hybridize to complementary sequences on the DNA chip, and the resulting DNA chip may be analyzed to determine which oligonucleotide probes have been hybridized. Analysis of the DNA chip may be performed by biotinylating the nucleic acids isolated from the tissue and fluid samples; once the nucleic acids are hybridized to the DNA chip, streptavidin coupled to a fluorescent dye may be added. Alternatively, streptavidin may be added, followed by staining with an anti-streptavidin antibody. The anti- streptavidin antibody may be conjugated to a fluorescent dye, or may be bound by an additional antibody which is conjugated to a fluorescent dye. The resulting fluorescence-stained DNA chip may be scanned with a confocal laser, which causes the fluorescent dye to fluoresce. The resulting fluorescence pattern may be used to determine which oligonucleotide probes have been hybridized. In a preferred embodiment, GENECHIPs™ may be used to determine expression.

Detecting the amplification products may include any step that detects specific hybridization of the isolated nucleic acids to one or more probe sequences. If a labeled probe hybridizes to the isolated nucleic acid, the label is preferably one that can be detected in a homogeneous system (i.e., one that does not require unbound probe to be separated from the isolated nucleic acid hybridized to probe for detection of bound probes). Alternatively, isolated nucleic acids or fragments thereof may be hybridized to an array of probes as on a DNA chip and those probes that specifically hybridize to the isolated nucleic acids are detected to provide sequence information about the isolated nucleic acids. Those skilled in the art will appreciate that more than one procedure may be used to detect the isolated nucleic acids.

5.2.2. Statistical Analysis Of Genetic Screening Data

Genetic screening data obtained utilizing the methods described above may be analyzed via well known statistical methods. Any method of statistical analysis which is well known in the art may be utilized in the present invention. In a preferred embodiment, ANOVA (analysis of variations) is utilized. The analysis may be corrected for multiple comparisons, utilizing, for example, the Benjamini-

Hochberg correction. Computer software may be utilized to perform the statistical analysis. Examples of computer software which may be used in the present invention include GENESPRING™ (Agilent Technologies, Palo Alto, California) and STATCRUNCH™ (Integrated Analytics). Utilizing statistical analysis techniques to compare data, a t-test may be performed to compare the subject sample to the control sample and to determine if differences in value are due to random fluctuations or are due to other contributing factors. Differences in value represent increases or decreases in gene expression, and whether they are due to random fluctuations or other contributing factors will depend upon the p-value derived. A p- value may be derived from the t-test results, and is a measure of the probability that increases or decreases in gene expression are due to random variations. Thus, a larger p-value indicates a greater likelihood that increases or decreases are most likely due to random variation; a smaller p-value indicates that increases or decreases are less likely to be random, and are caused by another contributing factor, such as genetic dysregulation due to IUGR. As used herein, an increase or decrease in gene expression is "statistically significant" if the p-value for the increase or decrease, relative to gene expression in a healthy subject, is less than or equal to 0.1, less than or equal to 0.05, or less than or equal to 0.01. Other methods of statistical analysis are well known in the art, and may also be performed. For example, linear regression may be performed as function of gene expression versus gestational age. Linear regression and other statistical analysis methods are well known in the art, and well within the capabilities of those of ordinary skill in the art.

5.2.3. Imprinted Genes

As noted above, genes subject to parental imprinting are interesting candidates for a role in IUGR, as they are known to suppress or promote fetal growth. Methods of identifying parentally imprinted genes and determining whether they are paternally or maternally imprinted are well known in the art. See, for example, Morison et al, Blood, 2000, 96(9):3023-3028. Many genes are known to be parentally imprinted, and persons of ordinary skill in the art can identify genes known to be parentally imprinted. See Morison et al, Human Molecular Genetics, 1998, 7(10): 1599- 1609. Non-limiting examples of maternally and paternally imprinted genes are included in Reik & Walter, Nature Genetics, 2001, 27:255-256. Imprinted genes can be identified using the methods identified in these references, or any other method that is known in the art.

Maternally expressed and paternally repressed genes include, but are not limited to: TP73, COMMDl , IGF2R, SLC22A2, SLC22A3, CALCR, PPP1R9A, PON2, PON3, ASB4, DLX5, CP A4, STOXl, CTNNA3, H19, ASCL2, PHEMX, CD81, TSSC4, KCNQl, KCNQlDN, CDKNlC, SLC22A1LS, SLC22A18, PHLDA2, NAP1L4, OSBPL5, ZNF215, DCN, HTR2A, MEG3, miR-337, Anti- PEGl I₅ MEG8, UBE3A, ATPlOA, GATM₅ TCEB3C, ZIM3, GNAS, TSIX. Other maternally expressed genes can be identified using the methods identified in these references, or any other method that is known in the art. Paternally expressed and maternally repressed genes include, but are not limited to: DIRAS3, NAP1L5, HYMAI, PLAGLl, SGCE, PEGlO, PONl, MEST₅ MESTITl, COPG2IT1, INPP5F, IGF2, IGF2AS, INS, TRPM5, KCNQlOTl, WTl-AIt transcript, WTlAS, SDHD, SLC38A4, DLKl, DLKl downstream transcripts, LOC388015, DIO3, MKRN3, ZNF127AS, MAGEL2, NDN, SNURF- SNRPN, GABRB3, GABRA5, GABRG3, RASGRFl, IMPACT, IMPOl, ITUPl, PEG3, USP29, ZNF264, NNAT, L3MBTL, SANG, XIST. Other paternally expressed genes can be identified using the methods identified in these references, or any other method that is known in the art.

5.2.3. Intrauterine Growth Restriction Related Genes

As used herein, "IUGR-related genes," "intrauterine growth restriction-related genes," "IUGR-associated genes," or "intrauterine growth restriction-associated genes" refer to genes which possess differential expression in fetuses or newborn infants with IUGR, as compared to normal fetuses or newborn infants which do not have IUGR. Genes identified by the methods disclosed in the present invention to be IUGR-related genes may be useful for the diagnosis, treatment, or prevention of IUGR. Genes identified to be IUGR-related genes may be used as diagnostic targets for the early detection and diagnosis of IUGR. IUGR- related genes may be targets for genetic therapy. IUGR-related genes may also be targets for agents which modulate their expression, for the treatment or prevention of IUGR.

The difference in gene expression in IUGR samples and non-IUGR is referred to as the "expression ratio," and can be expressed as a ratio between the gene expression of the IUGR samples and the non-IUGR samples.

(Gene Expression of Target Gene in IUGR Sample)

(Expression ratio) =

(Gene Expression of Target Gene in non-IUGR Sample)

The expression ratio represents the fold difference between IUGR and non-IUGR gene expression. Differential expression of genes is defined as when the expression ratio is increased or decreased by a statistically significant amount, wherein statistically significant refers to a p-value of 0.1 or less, 0.05 or less, or 0.01 or less. Whether a particular gene has increased or decreased expression will vary based upon the gene under study. An expression ratio of one (1) indicates that the gene expression in the IUGR samples and the non-IUGR samples are the same, and therefore the gene under study is not dysregulated by IUGR. Differential expression of genes where the expression ratio is less than 1, and wherein the decrease is statistically significant, indicates reduced expression of the target gene in the IUGR sample, as compared to the gene expression in a healthy, non-IUGR sample. Target genes which have reduced gene expression may be inhibited by IUGR, and accordingly may be a desirable target for the treatment or prevention of IUGR. Differential expression of genes where the expression ratio is greater than 1, and wherein the increase is statistically significant, indicates increased expression of the target gene in the IUGR sample, as compared to the gene expression in a healthy, non-IUGR sample. Target genes which have increased gene expression may be activated by IUGR, and accordingly may be a desirable target for the treatment or prevention of IUGR. Gene expression is preferably measured with multiple samples, in order to ensure a sufficient sampling size and to increase the confidence level in the statistical analysis.

Examples of genes which have increased or decreased gene expression, and wherein the increase or decrease in expression is statistically significant, can be found in Tables 2 and 3.

For genes which display an increase in expression, the expression ratio may be at least 1.25, at least 1.5, at least 2.0, at least 4.0, at least 7.5, or at least 10.0. Statistical analysis may be performed to determine whether the increase in expression ratio is statistically significant. Genes which manifest increased expression of in

IUGR, i.e., have expression ratios which are greater than 1, include but are not limited to: ADAM12, ADAM19, ENTPDl, LGALS14, PROCR, PTPRF, RAI, SDCl, SSF A2, TFRC, ALPP, CGA, CRH, FBLNl, GDF15, GH2, INHBA, LEP, MFAP5, PAPPA, PLAC3, PRSSI l, PSGl, PSG3, PSG4, PSG9, TFPI, TFPI2, TIMP2, TUFTl, 7h3, ABCGl₅ ABHD5, ACSL4, ADFP, ADK, AMDl, BCAR3, BCL6, BZW2, C14orf58, C6orf4, CAP2, CAPN6, CEBPB, CLTB, CMAH, COBLLl, CRIPl, CSF2RB, CYB5R1, CYP19A1, DKFZP564O123, EBI3, EFHDl, FDXl, FLJlOl 16, FLJ14146, FLJ38507, FOXJ3, FTS, GABARAPLl, GATA3, GLRX, GULPl, HEXB, HISTlHlC₅ HMGB3, HOP₅ HPGD, HSD3B1, HSPBl, HTATIP2, ICK₅ IDS₅ IER5, IGSF3, ILlRAP₅ INSIGl₅ KIAA0626, KIAAl 102, KIBRA₅ KIF2, LIMK2, LOC90333, LRRCl₅ MAFF₅ MANlCl, MOBKL2B, NUCB2, OAZIN₅ P24B, PDLIM2, PGRMC2, PLCL2, PRKAG2, RASGRPl, RFK₅ SlOOP₅ SAT₅ SIATlO₅ SLCl 1A2, SLC16A3, SLC39A8, SLC7A1, SLCO2A1, SMARCBl₅ SNXlO, STK3, TCF4, TFAP2A, THEDCl, TJP2, TNKS2, ZCCHC2.

For genes which display a decrease in expression, the expression ratio may be at most 0.9, at most 0.7, or at most 0.5, at most 0.3, or at most 0.1. Statistical analysis may be performed to determine whether the decrease in expression is statistically significant. Genes which manifest decreased expression of in IUGR₅ i.e., have expression ratios which are less than I₅ include but are not limited to: ADAMTSL3, ClR₅ CD44, DKFZP586H2123, OLFML3, PTPRD₅ PTPRK₅ SLIT2, SRPX₅ STABl, THSDl, THYl, Z39IG, AOC3, BMP5, COLl 4Al, COL15A1, COL21A1, COL5A2, COL6A1, COL6A3, DEFAl, ENPP2, FMOD, HG4, IGFl₅ LAMA2, LAMC3 , LIPG, LOXL2, MMP2, NID 1 , PCOLCE₅ WFDC 1 , WNT2, AGTRl₅ CBFA2T1, CHClL, CHNl, CNN3, CYBB₅ DOCl₅ DPYSL3, DSCRl₅ EBIl₅ ENPPl₅ EPB41L2, FCERlG, FCRlA, FLJ10652, FLJl 1175, FLJ22344, GPR124, PUCY1A3, GUCY1B3, ICAM2, IFIl 6, ITGAl, KCNK3, KCNS3, KL, LAPTM4B, LEFl, LNK, LOC221362, LPHN2, LYRIC, MEF2C, MEG3, MEISl, MYOlO, MYOlB, NR3C1, PDZRN3, PMP22, SNAI2, SPRYl₅ TCF4, TGFBlIl₅ ZFHXlB.

The present invention also encompasses oligonucleotide sequences which are complementary to IUGR-related genes. The oligonucleotides may be utilized as primers for amplification of the IUGR-related genes, for example, by polymerase chain reaction (PCR). The oligonucleotides may also be utilized as probes for the detection of IUGR-related genes. Preparation of primers or probes using well-known methods based upon IUGR-related genes will be readily apparent to those of ordinary skill in the art. Examples of IUGR-related genes are included in Tables 2 and 3. 5.3. Kits for Diagnosing Intrauterine Growth Restriction

The present invention further provides kits for diagnosing IUGR in a subject. The methods, PCR primers, and nucleotide sequences described herein may be efficiently utilized in the assembly of a diagnostic kit, which may be used to diagnose IUGR in a subject. The kit is useful in distinguishing between fetuses or newborn infants with IUGR or mothers carrying fetuses with IUGR, and normal, healthy fetuses or newborn infants or mothers bearing normal, healthy fetuses. Such a diagnostic kit contains the components necessary to practice the methods as described above.

Thus, the kit may contain a sufficient amount of at least one probe complementary to an IUGR-related gene. The kit may also contain a sufficient amount of at least one PCR primer pair for an IUGR-related gene, for the amplification of the IUGR-related gene. The kit may optionally comprise components of a detectable labeling system, vials for containing the tissue or fluid samples, control tissue or fluid samples (e.g., dried or frozen tissue or fluid from a healthy fetus, newborn infant, or mother, or preparations containing nucleic acids, proteins, or other compounds which may represent the normal samples), protein samples, and the like. Other conventional components of such diagnostic kits may also be included. The oligonucleotide probes may be complementary to the sequences identified in Table 2. In a preferred embodiment, the oligonucleotide probes comprise sequences complementary to portions of PHLD A2 or MEST. The kit may comprise an oligonucleotide probe directed to a non-IUGR related gene; said probe may be a hybridization probe or a PCR primer. Alternatively, the kit may contain a sufficient amount of at least one primer pair or probe complementary to a maternally expressed/paternally repressed IUGR-related imprinted gene, and at least one primer pair or probe complementary to a maternally repressed/paternally expressed IUGR-related imprinted gene. The kit may optionally comprise components of a detectable labeling system, vials for containing the tissue or fluid samples, control tissue or fluid samples (e.g., dried or frozen tissue or fluid from a healthy fetus, newborn infant, mother, or preparations containing nucleic acids, proteins, or other compounds which may represent the normal samples), protein samples, and the like. Other conventional components of such diagnostic kits may also be included.

The diagnostic kits may also include instructions for using the included components. The instructions may also include methods of calculating the ratio of IUGR gene expression to non-IUGR gene expression. The instructions may include methods of calculating the ratios of paternally expressed/maternally repressed genes to paternally repressed/maternally expressed genes. The instructions may include charts, which may be visual or textual in nature, to aid in the interpretation of gene expression ratios. The kit may also include computer software to aid in the measurement of gene expression or the calculation of expression ratios. The kit may also include or provide access to a database which contains normalized gene expression values derived from healthy subjects without intrauterine growth restriction for one or more IUGR related gene(s).

The kits may additionally comprise reagents and equipment for purifying nucleic acids from tissue or fluid samples, which may include any reagents or equipment known to persons of ordinary skill in the art for purification of nucleic acids. In a preferred embodiment, the reagent for purifying RNA from a tissue or fluid sample is TRIZOL™. Reagents and equipment for measuring gene expression may include any reagents or equipment known to persons of ordinary skill in the art for detecting gene expression. In a preferred embodiment, the reagents and equipment for measuring gene expression includes a GENECHIP™. The GENECHIP™ may be constructed to detect expression of one or more of the gene sequences identified in Table 2. In a preferred embodiment, the GENECHIP™ is constructed to detect PHLD A2 and MEST genes. Control reagents may comprise healthy tissue samples, or tissue or fluid samples which have known expression levels for particular genes. The control reagents may be fresh, frozen, or otherwise preserved.

The kit for diagnosing IUGR may comprise: (1) oligonucleotide probes directed to intrauterine growth restriction related genes; (2) reagents and equipment for measuring gene expression; and (3) control reagents. Other known assay formats will indicate the inclusion of additional components for a diagnostic kit according to this invention. For example, the reagents and equipment for purifying nucleic acids from tissue or fluid samples may include oligonucleotide probes for use in identifying or amplifying the presence of particular genes.

The kit for diagnosing IUGR may include oligonucleotide probes comprising sequences complementary to portions of the following genes: CD 81 (Genbank Accession No. NM_004356), CDKNlC (Genbank Accession No. NMJ)00076), DCN (Genbank Accession No. NMJ 73906), DIO3 (Genbank Accession No. NM_001362), DLKl (Genbank Accession No. NM_001032997), GATM (Genbank Accession No. NM_001482), GNAS (Genbank Accession No. NMJ)00516), GRBlO (Genbank Accession No. NMJ)01001549), HYMAI (Genbank Accession No. AF241534), IGF2 (Genbank Accession No. NM _000612), IGF2R

(Genbank Accession No. NM_000876), MEG3 (Genbank Accession No. AFl 19863), MEST (Genbank Accession No. NM_002402), MKRNl (Genbank Accession No. NMJ) 13446), NDN (Genbank Accession No. NM_002487), NNAT (Genbank Accession No. NM_005386), PEG3 (Genbank Accession No. NM_006210), PLAGLl (Genbank Accession No. NM _002656), PON2 (Genbank Accession No.

NMJ)00305), PPPlCC (Genbank Accession No. NM_002710), SGCE (Genbank Accession No. NM_003919), SNRPN (Genbank Accession No. NM_003097), SNURF (Genbank Accession No. NM_005678), PHLD A2 (Genbank Accession No. NMJ)O3311), UBE3A (Genbank Accession No. NM_000462), ZIM2 (Genbank Accession No. NMJ)15363), AGTRl (Genbank Accession No. NM_004835, NMJ)00685), CRH (Genbank Accession No. NM_000756, BC002599), DSCRl (NM_004414, AL049369), GLRX (Genbank Accession No. NM_002064, AF162769), HPGD (Genbank Accession No. NM_000860, J05594, U63296), IGFl (Genbank Accession No. Al 078169, M29644), INDO (Genbank Accession No. M13436), INHBA (Genbank Accession No. M13436), LEP (Genbank Accession No. NMJ)00230), PSG4 (Genbank Accession No. NM_002780, NM_006905, NMJ)02783), or functionally equivalent genes. Other oligonucleotide probes may be included which are complementary to the sequences identified in Tables 2 and 3. The following nonlimiting examples serve to further illustrate the present invention.

6. EXAMPLES

Materials and methods Subjects and placental tissues Samples were derived from excess tissue from existing placenta specimens received for pathologic diagnosis. A 0.8 cm cube of placental tissue was excised immediately below the fetal surface in each case and either snap-frozen or preserved in RNALATER™ reagent (Ambion, Austin, Texas). In some cases three quadrants of a single placenta were sampled. Gestational age, placental weight and birth weight were recorded, and clinical information was obtained from Doppler ultrasound examination in cases of suspected IUGR. Each placenta received a complete assessment of histopathology. In addition, four placentae were obtained from pregnancies with severe IUGR from the University of Toronto, using a similar procedure for tissue procurement.

Northern and Southern blotting

RNA from placental tissues pulverized under liquid nitrogen was prepared using TRIZOL™ (Invitrogen) and was resolved on formaldehyde-containing agarose gels and transferred to Nytran membranes (Schleicher and Schull). Northern blotting probes for PHLD A2, MEG3 , MEST and GAPDH were partial cDNAs prepared by RT-PCR using gene-specific primers (sequences available on request). Hybridization with the 32P-labeled probes was in ULTRAhyb (Ambion) at 42 ⁰C overnight; washing was at 64 ⁰C in 0.1% SDS/0.1 x SSC. Band intensities on the autoradiograms were measured with a Storm Phosphorimager (Model 840, Molecular Dynamics/ Amersham). Genomic DNA was prepared by SDS/Proteinase K lysis followed by phenol/chloroform extraction and ethanol precipitation. The purified DNA, 4 μg, was digested with the indicated restriction enzymes overnight, followed by electrophoresis through 1% agarose gels, denaturation/neutralization, and transfer to Nytran membranes. Hybridization and washing were as described above. The KvDMRl probe was a genomic fragment synthesized by PCR using the primers KvDMRl US (CAGGCAGCAGAAAACAAAACAGAG) and KvDMRl DS (TTAGAGGTCTCAGTGGGGTATGGG).

Microarray analysis and statistical methods

HG-Ul 33 A GeneChips (Affymetrix) were used to analyze human placental RNA. The cRNA probes were synthesized as described previously (Li et al, 2002; Li et al. 2004). After scanning the chips, the fluorescence intensities for each probe set were determined using Affymetrix GeneChip Software, and the intensity data were pre-processed to maximize linearity using the Robust Multi- Array Analysis (RMA) algorithm in the GeneSpring software package (Silicon Genetics). Using GeneSpring, the expression values for each probe set were first filtered for raw signal (>250 in at least four samples) and variation (1.2-fold deviation from the experiment mean in at least four samples), and genes (probe sets) that passed these criteria were subjected to ANOVA, using the Cross-Gene Error Model of GeneSpring, and the

Benjamini-Hochberg correction for multiple comparisons, to obtain sets of genes that differed in their expression between the IUGR and non-IUGR placentae. Dendrograms were created using the GeneTree function of GeneSpring. For multiple linear regression analysis a Web-based tool available at http://www.statcrunch.comhttp://www.statcrunch.com was used. Immunohistochemistry

An affinity-purified rabbit polyclonal antibody (C-134) raised against a synthetic peptide whose amino acid sequence was derived from the C-terminal portion of IPL was used at a 1 :1000 dilution as described previously (Saxena et al. 2003) with the following modifications: antigen retrieval was carried out in 1 mM EDTA by boiling slides in a 1000 W microwave oven for 8 min at 100% power followed by 15 min at 30% power; no blocking of endogenous biotin was performed, and goat anti-rabbit secondary antibody was used at 1 :200 dilution, following the protocol of the Vectastain Elite ABC kit and Nova Red substrate (Vector Labs). Results

Increased PHLDA2/MEST mRNA ratio determined by Northern blotting in IUGR compared to non-IUGR placentae

The initial analysis focused on two oppositely imprinted genes: the maternally expressed/paternally repressed gene PHLDA2 (a.k.a. IPL, TSSC3, BWRlC) encoding a small cytoplasmic protein with a pleckstrin-homology domain, and the paternally expressed/maternally repressed gene MEST (a.k.a. PEGl) encoding a hydrolase enzyme with unknown substrates. These two genes have opposite effects on placental growth in mice, with knockout experiments showing that Phlda2 restrains growth (Frank et al. 2002 and Salas et al. 2004) and Mest promotes it (Lefebvre et al. 1998). Both of these genes are expressed at high levels in cytotrophoblast (Saxena et al. 2003 and Mayer et al. 2000), and can be examined simultaneously on Northern blots of total RNA from human placentae. The ratio of mRNA from these two oppositely imprinted genes was assessed, comparing IUGR to non-IUGR placentae. The initial case series consisted of 38 IUGR-associated placentae and 75 non-IUGR placentae with a similar range of gestational ages. IUGR in this series was defined as neonatal birth weight below the 10th percentile for gestational age, relative to a United States reference (Oken et a 2003). Cases of preeclampsia were not excluded from this series. Overall, the mean placental weight was significantly lower, birth weight significantly lower and gestational age slightly less (not significant) among the cases with IUGR compared to those without IUGR (Table 1). Total RNA and DNA was extracted from the tissue and measured PHLD A2 and MEST mRNA levels by Northern blotting and Phosphorimaging. The procedure consisted of a series of hybridizations and exposures of the blots, first with the individual PHLD A2 and MEST probes, each consisting of a partial cDNA (primers for probe synthesis available on request), and then with a 1 :1 mixture of these probes (examples in Figure 1). The blots were stripped between hybridizations. Band intensities and ratios were calculated by Phosphorimaging after subtracting the background value for each lane. As a control for spatial variation in gene expression within a given placenta, four non-IUGR placentae were analyzed, each sampled from three different cotyledons. Minimal variation was found in the PHLDA2/MEST mRNA ratio in each case (Figure 1 and data not shown).

As shown in Figure 2 and Table 1, the PHLDA2/MEST mRNA ratio was significantly higher in the IUGR-associated placentae compared to those without IUGR (mean 0.49 in non-IUGR; 0.71 in IUGR, p = 0.001), and as shown in Figure 2 this effect was evidently driven by a cluster of IUGR cases with high PHLDA2/MEST mRNA ratios. There was a significant negative correlation between the PHLDA2/MEST mRNA ratio and placental weight (r = -0.35, p = 0.001), and a univariate analysis of variance with the PHLDA2/MEST mRNA ratio as the dependent variable showed that the increase in this ratio among the IUGR-associated placentae persisted after adjusting for placental weight and gestational age (F for IUGR of 8.47, p = 0.004).

While many cases of IUGR are idiopathic, fetal and placental growth restriction can also result from defined causes, including multiple gestations, and fetal developmental anomalies. Such factors, which in themselves can account for the IUGR, might not be expected to correlate with altered expression of imprinted genes. Available in this series, but excluded from the statistical analysis, were two cases of IUGR associated with severe fetal anomalies (one with cardiac malformations and one with gastroschisis), and three cases of IUGR in twin pregnancies. As shown in Figure 2, all five of these cases showed low PHLDA2/MEST mRNA ratios. It was also investigated whether preeclampsia, a common pregnancy complication that is distinct from IUGR, might be associated with an altered PHLDA2/MEST mRNA ratio. Among the pregnancies, 18 carried a clinical diagnosis of preeclampsia. The mean PHLDA2/MEST mRNA ratio was 0.54 in the absence of preeclampsia, and was slightly increased at 0.64 in the placentae associated with preeclampsia (p = 0.28, not significant). Thus, while preeclampsia was somewhat more common among the cases with IUGR (22.9%) compared to without IUGR (11.8%), it was not independently associated with an increased PHLDA2/MEST mRNA ratio.

PHLDA2 and MEST were also assessed individually, normalizing by reference to the Northern blot band intensities obtained with a GAPDH "housekeeping" gene probe. The PHLDA2/GAPDH mRNA ratio was increased in the IUGR-associated placentae overall (mean 0.29 in non-IUGR; 0.38 in IUGR), although this trend did not reach statistical significance (p = 0.088, T-test), and there was a significant decrease in the MEST/GAPDH ratio in the IUGR-associated placentae (mean 0.24 in non-IUGR; 0.14 in IUGR, p = 0.004, T-test).

Lack of altered DNA methylation at the imprinting centers of PHLDA2 and MEST

DNA methylation was next assessed at the well-studied differentially methylated region (DMR) linked to the PHLD A2 gene - the KvDMRl /LITl element on chromosome 1 IpI 5.5. Southern blotting of genomic DNAs digested with methylation-sensitive restriction enzymes revealed a normal pattern of methylated and non-methylated bands of equal intensity (representing the imprinted and non- imprinted alleles) in seven IUGR-associated placentae that had shown high PHLDA2/MEST mRNA ratios (>0.75) by Northern blotting (Figure 3 and data not shown). As a positive control for altered DNA methylation in this sequence genomic placental DNA from a case of Beckwith-Wiedemann syndrome (BWS) with placentomegaly was used. This showed loss of the upper band on the Southern blot - a diagnostic pattern for loss-of-imprinting in this disorder. Using a similar Southern blotting approach (McMinn et al.), the major DMR situated at the 5' end of the MEST gene was, and no abnormality was found in its DNA methylation (data not shown). It is concluded that the high PHLDA2/MEST mRNA ratios in this subset of IUGR may reflect altered DNA methylation in as yet uncharacterized cis-acting regulatory sequences, but more likely reflects conventional transcriptional dysregulation by trans-acting factors in placental cytotrophoblast. Immunohistochemical analysis of PHLD A2 protein in IUGR placentae

As previously reported, PHLD A2 protein is easily detectable in villous cytotrophoblast of normal human placentae by immunostaining of formalin-fixed paraffin-embedded tissue with an affinity-purified polyclonal anti-PHLDA2 (IPL) antiserum (Saxena et al. 2003 and Thaker et al. 2004). This method was used to determine whether the distribution and intensity of PHLD A2 immunoreactivity was altered in five cases of IUGR with changes of maternal vascular under-perfusion, compared to four age-matched normal placentae. As shown in Figure 4, the staining within the villi was limited to the villous cytotrophoblast cells in both groups of cases. PHLDA2 immunostaining was also weakly positive in the extravillous (intermediate) trophoblast in both groups. Consistent with the observed increase in PHLD A2 mRNA, PHLDA2-positive cytotrophoblast appeared to be more strongly stained in cases of IUGR, compared to normal placentae. However, immunohistochemistry is not a quantitative method for measuring protein expression, and the major conclusion from examining these tissue sections is that PHLD A2 protein remains appropriately cell type-specific in IUGR placentae. Immunostaining for MEST protein has not been performed, but this analysis, and immunostaining for the protein products of other dysregulated imprinted genes (see below) is of interest for future work.

A genome- wide survey reveals unbalanced expression of additional imprinted genes in IUGR placentae The data in Figure 2 was next more closely to ask whether any clinical or pathological feature might identify the cluster of cases of IUGR with high PHLDA2/MEST mRNA ratios. A review of the pathology reports indicated that 60% of the IUGR cases overall had histopathological evidence of maternal vascular under- perfusion as evidenced by presence of one or more of the following: increased syncytial knots, villous agglutination, hypermature villi, distal villous hypoplasia, increased intervillous fibrin, chronic infarcts and maternal decidual vasculopathy (Redline et al. 2004). But among the nine IUGR cases with PHLDA2/MEST mRNA ratios >1 by Northern analysis, eight (89%) showed such changes. The changes in expression of both imprinted and non-imprinted genes in this class of IUGR were more comprehensively assessed, using oligonucleotide microarrays. This approach has previously been validated with Affymetrix GENECHIPs™ in surveys of gene expression in normal and neoplastic human tissues, confirming the microarray data for >20 genes by Northern blotting, with no discordant results (Li et al. 2002 and Li et al 2004). 14 IUGR placentae were selected with histopathology indicative of maternal vascular under-perfusion: including 10 cases from the Columbia University and 4 cases obtained from the University of Toronto. In this series twin pregnancies and cases with severe fetal anomalies were excluded. As controls, 15 non-IUGR placentae from Columbia U. spanning this same range of gestational ages were used (mean GA 33.1 weeks in non- IUGR; 32.6 weeks in IUGR, T-test, not significant).

Affymetrix U133Av2 oligonucleotide microarrays were hybridized with cRNA probes from these cases. After first filtering the GeneChip data for minimum signal intensity (see Methods) ANOVA was carried out employing the

Benjamini-Hochberg correction for multiple comparisons at a false discovery rate of 0.05, and found 202 genes (245 Affymetrix probe sets) significantly over-expressed and 207 genes (250 Affymetrix probe sets) significantly under-expressed in the IUGR placentae (Table 3). In this analysis, 27 imprinted genes (CD81, CDKNlC, DCN, DIO3, DLKl, GATM, GNAS, GRBlO, HYMAI, IGF2, IGF2R, MEG3, MEST₅

MKRNl, NDN, NNAT, MESTO, PEG3, PLAGLl, PON2, PPPlCC, SGCE, SNRPN, SNURF, PHLD A2, UBE3A,and ZIM2) gave reliable signals in four or more samples and could therefore be evaluated for differential expression. Among these genes, PHLDA2 expression was increased and MEST, MEG3, GATM, GNAS and PLAGLl expression was decreased in IUGR. In addition, analysis of the GeneChip data by ANOVA without the Benjamini— Hochberg statistical correction showed that IGF2 mRNA was decreased and CDKNlC mRNA increased on average in the IUGR placentae, albeit less reliably (L e. with greater case-to-case variability) than the other differentially expressed imprinted genes (see T-test p-values for individual genes in Table 2 and the Table 3). These data for IGF2 and CDKNl C are nonetheless of interest given the genetically confirmed role of these two genes in regulating placental growth (Caspary et al. 1999, Takahashi et al 2000; Eggenschwiler et al 2002; Constancia et al. 2002; Baker et al. 1993; Lage et al. 1991; Drut et al. 1996; McCowan et al. 1994; and Reish et al. 2002).

Some genes are represented by more than one oligonucleotide probe set. The p-values are from multiple linear regressions, testing for differences in mRNA expression as a function of diagnosis (IUGR vs. non-IUGR), and then adjusting for gestational age.

Since the statistical approach in generating the Table 3 employed the Cross-Gene Error Model in GeneSpring, and the Benjamini-Hochberg correction for multiple comparisons, two genes shown here (IGF2, CDKNlC) do not appear in the Table 3.

Validation of the differential expression of MEG3 by Northern blotting Independent technical validations of microarray data are desirable, and as shown above, validation for PHLD A2 and MEST was done by Northern blotting. Validation for the imprinted MEG3 gene was also performed. Northern blotting in a series of 30 IUGR and 46 non-IUGR placentae for which ample RNA was available confirmed that placental MEG3 RNA is indeed reduced on average in IUGR (Figure 5a, b). Interestingly, there is an increase in MEG3 RNA in non-IUGR placentae close to term (Figure 5b), and the normal gestational age-dependence of its placental expression is evidently lost or diminished in IUGR (Figure 5b). Differential expression of non-imprinted genes in IUGR vs. non-IUGR placentae

The 202 genes over-expressed and 207 genes under-expressed in the complete series of 14 IUGR and 15 non-IUGR placentae by the Benjamini- Hochberg-corrected ANOVA with a false discovery rate of 0.05 are organized by the relatedness of their expression patterns across the 29 cases in the dendrogram in

Figure 6 and listed in the Table 3. Several classes of non-imprinted genes (or genes with unknown imprinting status) on these lists are of obvious interest. These include genes involved in endocrine signaling, notably those encoding corticotrophin releasing hormone (CRH), leptin (LEP), hydroxyprostaglandin dehydrogenase (HPGD), and the prostaglandin transporter (SLCO2A1), all of which were over- expressed on average in the IUGR cases. On the dendrogram, the CRH and HPGD genes, as well as the pregnancy-specific glycoprotein (PSG) genes, which encode immune-modulating proteins, appear in clades characterized by substantial case-to- case variability, with gene activation seen not only in IUGR but also in a minority of the non-IUGR cases (Figure 6). This pattern suggests that these genes might be activated by placental stresses that are more common in IUGR but are not unique to this condition. In fact, elevated corticotrophin releasing hormone has been previously associated both with small-for-gestational age pregnancies and premature labor

(Wadhwa et al. 2004). Also, in the category of endocrine signaling, the INHBA gene, encoding a TGF-beta superfamily member, inhibin beta-1, with roles in reproduction and development, was up-regulated on average in the placentae with IUGR. The gene for indoleamine-pyrrole 2,3-dioxygenase (INDO/IDO), which is an immune modulator essential for toleration of the fetal hemi-allograft in a mouse model system (Munn et al. 1998 and Mellor et al. 2001), was over-expressed in the IUGR placentae. Several growth factor genes were differentially expressed, including insulin-like growth factor-1 (IGFl), which like IGF2 (but with greater statistical significance) was under-expressed in IUGR. At least one gene dedicated to the response to oxidative damage (glutaredoxin, GLRX) was strongly and consistently up-regulated in the

IUGR cases (the thioredoxin gene, TXN, also showed increased mRNA in the IUGR placentae but did not pass the statistical cutoff in the combined series), and several metabolite transporter genes (SLC-family facilitated diffusion channels) were differentially expressed, with some family members over-expressed and others under- expressed in IUGR (Supplementary table). Two genes controlling vascular function, DSCRl, encoding a calcineurin inhibitor, and AGTRl, encoding the angiotensin II type I receptor, were consistently down-modulated in IUGR. The differences in mRNA expression for the above genes in IUGR vs. non-IUGR after adjusting for gestational age were also assessed, and all remained highly significant (Table 2). Discussion

Among the roughly 30 imprinted genes that have known biological functions, a large percentage are expressed in trophoblast and control placental growth and/or development (Tycko et al. 2002; Reik et al. 2003; and Weksberg et al. 2003). Moreover, genetic experiments in mice and phenotypes of human syndromes involving imprinted genes for the most part support the conflict hypothesis, which states that maternally expressed genes such as Phlda2 and Cdknlc restrict fetal and placental growth to the energetic advantage of the mother, while paternally expressed genes such as Mest and Igf2 augment fetal and placental development at the expense of maternal resources (Tycko et al. 2002 and Haig et al. 1996). This evidence for imprinted genes as one possible point of regulation for placental growth provided motivation for testing the hypothesis that imprinted genes in the human placenta might be dysregulated in IUGR, and the results described here suggest that this is true. Given the known functions of the murine Phlda2 and Mest genes in negatively and positively regulating placental growth, respectively, the finding that the PHLDA2/MEST mRNA ratio is increased in placentae from cases of human IUGR suggests that the dysregulation of these two imprinted genes (and certain other genes, see below) may be an adverse response to the underlying pathophysiology of IUGR, which is often maternal vascular under-perfusion of the placenta. That is, the placenta may respond to chronic hypoperfusion by activating a program of gene expression that further restricts placental growth. This response would be adverse, perpetuating a vicious cycle of growth restriction, in situations in which the entire placenta is poorly perfused, as in IUGR. However, this same type of transcriptional response might be physiologically advantageous when the hypoperfusion is only regional within an otherwise normal placenta.

As a technical point, IUGR can have various etiologies, and gene expression in the placenta will likely vary among these different classes of growth restriction. Sadovsky and colleagues recently found that PHLD A2 (BWRlC in that study) was down-regulated in growth-restricted placentae from twins that were discordant for IUGR (Roh et al. 2005). The data presented here does not contradict this result, since it was also found that low PHLDA2/MEST mRNA ratios in the IUGR cases associated with twinning.

In addition to PHLD A2 and MEST, several of the other imprinted and non-imprinted genes that were differentially expressed as a function of IUGR in the microarray data are biologically interesting. MEG3 (a.k.a. Gtl2 in mice) is a maternally expressed gene that produces a non-coding RNA which was found to be expressed at reduced levels in the IUGR placentae. MEG3 is located in an imprinted domain on human chromosome 14/mouse chromosome 12, and while the specific function of MEG3 RNA is unknown, the phenotypes of uniparental disomies have implicated this overall chromosomal region in fetal and placental growth(Robinson et al. 1996; Georgiades et al. 2000; and Kotzot et al. 2004). The GATM gene encodes an enzyme in creatine synthesis, and this gene, which was under-expressed in the IUGR placentae in the series, is imprinted in the mouse placenta (Sandell et al. 2003). The biological significance of GATM down-modulation in human IUGR is uncertain, but placentomegaly resulting from paternal UPD at mouse chromosome 2 suggests that the murine orthologue, Gatm, is a candidate for growth restriction in the placenta (Cattanach et al. 2004). GNAS is a complex imprinted locus involved in metabolic regulation and the importance of its reduced expression in IUGR is not immediately clear. PLAGLl (a.k.a. ZAC) encodes a DNA-binding protein. This gene shows conserved imprinting in humans and mice, but data on its role in controlling placental growth are not yet available. The IGF2 gene, encoding insulin-like growth factor-2, has a clear role in supporting placental and fetal growth, and the reduced, albeit variable, expression of this gene in IUGR placentae may perpetuate the placental growth deficiency, consistent with the adverse cycle scenario described above. The CDKNlC gene, encoding a cyclin-cdk inhibitor, is closely linked to PHLD A2 in chromosome band 1 Ipl5.5, imprinted in the same direction (maternal allele active; paternal allele repressed), and controlled by the same cis-acting imprinting center, the KvDMRl element (Fitzpatrick et al. 2002). Like PHLDA2, but with lower statistical significance, CDKNlC mRNA was increased on average in the IUGR placentae in the series, and since p57CDKNlC inhibits cell proliferation, this finding is also consistent with the adverse cycle hypothesis. Not surprisingly, many other genes, non-imprinted or of unknown imprinting status, showed strong and consistent differences in their mean mRNA levels in IUGR vs. non-IUGR placentae (Table 2 and Table 3). These included genes involved in endocrine signaling, notably CRH, LEP and HPGD, as well as the prostaglandin transporter gene SLCO2A1, which were strongly over-expressed in the IUGR cases. As mentioned above, CRH and HPGD showed expression patterns among the cases that suggested a secondary involvement in IUGR, since these genes were sometimes highly expressed in non-IUGR pre-term placentae. The leptin gene (LEP) was a somewhat more specific marker for the IUGR placentae in the microarray data, but leptin expression is also dysregulated in preeclampsia (Poston et al. 2002). Up-regulation of the glutaredoxin (GLRX) gene in the IUGR placentae may reflect a response to oxidative stress (Jauniaux et al. 2005), and since the altered expression of oxidative stress-responsive genes in placental diseases already seems controversial (Shibata et al 2001; Sahlin et al. 2000; Takagi et al. 2004), it will be interesting to follow-up these observations in future studies. Since transporters in the placenta are a direct link between the mother and the fetus (Reik et a 2003), genes encoding metabolite transporters are of obvious interest in IUGR. The mRNAs for several SLC (major facilitator family) metabolite transporters, including amino acid and ion transporters, showed strongly differential expression in the IUGR vs. non- IUGR placentae (Table 3). Since maternal vascular under-perfusion is a major contributing factor in IUGR, genes involved in vascular function are also of interest. AGTRl and DSCRl are two such genes (Benetos et al. 1996; Nalogowska-Glosnicka et a 2000; Takahashie? ah, Ann Hum Genet 2000; Hesser et a 2004; Yao et ah 2004), and their under-expression in IUGR placentae may affect placental perfusion. Other genes potentially involved in vascular development or function, such as angiopoietin-like-2 (ANGPTL2), also appear on the list of differentially expressed genes (Table 3). Lastly, several non-imprinted growth factor genes were found to be differentially expressed, including insulin-like growth factor- 1 (IGFl), which was more consistently down-modulated than IGF2 (Table 2).

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[42] L. Poston, Leptin and preeclampsia, Semin Reprod Med 20 (2002), pp. 131-138. [43] E. Jauniaux and GJ. Burton, Pathophysiology of histological changes in early pregnancy loss, Placenta 26 (2005), pp. 114-123. [44] E. Shibata, K. Ejima, H. Nanri, N. Toki, C. Koyama and M. Ikeda et al, Enhanced protein levels of protein thiol/disulphide oxidoreductases in placentae from pre-eclamptic subjects, Placenta 22 (2001), pp. 566-572. [45] L. Sahlin, E. Ostlund, H. Wang, A. Holmgren and G. Fried, Decreased expression of thioredoxin and glutaredoxin in placentae from pregnancies with preeclampsia and intrauterine growth restriction, Placenta 21 (2000), pp. 603-609. [46] Y. Takagi, T. Nikaido, T. Toki, N. Kita, M. Kanai and T. Ashida et al, Levels of oxidative stress and redox-related molecules in the placenta in preeclampsia and fetal growth restriction, Virchows Arch 444 (2004), pp. 49-55. [47] A. Benetos, S. Gautier, S. Ricard, J. Topouchian, R. Asmar and O. Poirier et al, Influence of angiotensin-converting enzyme and angiotensin II type 1 receptor gene polymorphisms on aortic stiffness in normotensive and hypertensive patients, Circulation 94 (1996), pp. 698-703. [48] K. Nalogowska-Glosnicka, B.I. Lacka, MJ. Zychma, W. Grzeszczak, E. Zukowska-Szczechowska and R. Poreba et al., Angiotensin II type 1 receptor gene Al 166C polymorphism is associated with the increased risk of pregnancy-induced hypertension, Med Sci Monit 6 (2000), pp. 523-529. [49] N. Takahashi, H. Murakami, K. Kodama, F. Kasagi, M. Yamada and T. Nishishita et al., Association of a polymorphism at the 5 '-region of the angiotensin II type 1 receptor with hypertension, Ann Hum Genet 64 (2000), pp. 197-205.

[50] B.A. Hesser, X.H. Liang, G. Camenisch, S. Yang, D.A. Lewin and R. Scheller et ah, Down syndrome critical region protein 1 (DSCRl), a novel VEGF target gene that regulates expression of inflammatory markers on activated endothelial cells, Blood 104 (2004), pp. 149-158. [51] Y.G. Yao and E.J. Duh, VEGF selectively induces Down syndrome critical region 1 gene expression in endothelial cells: a mechanism for feedback regulation of angiogenesis?, Biochem Biophys Res Commun 321 (2004), pp. 648-656. [52] D. Dao, CP. Walsh, L. Yuan, D. Gorelov, L. Feng and T. Hensle et al, Multipoint analysis of human chromosome I lpl5/mouse distal chromosome 7: inclusion of H19/IGF2 in the minimal WT2 region, gene specificity of Hl 9 silencing in Wilms' tumorigenesis and methylation hyper-dependence of Hl 9 imprinting, Hum MoI Genet 8 (1999), pp. 1337-1352. [53] Poon et ah, Presence of Fetal RNA in Maternal Plasma, Clinical Chemistry, 2000, 26(11):1832-1834.

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Various references are cited herein, which are hereby incorporated by reference in their entireties.

Table 1. Parameters in pregnancies with and without IUGR included in the PHLDA2/MEST~Noήhem blotting series

Without IUGR IUGR

Sample size 75 38

PHLDA2/MEST mRNA 0.487 (0.215) 0.714 (0.464) ratio in placenta (mean ± S. D.)*

Placenta weight (mean ± S.D.)** 419.5 (162.1) 287.8 (103.6)

Gestational age (mean ± S.D.)*** 36.1 (5.3) 35.4 (4.0)

Birth weight (mean ± S.D.)**** 2655.9 (1136.5) 1795.9 (753.8) r-tests: *p = 0.0001 ; **p = 0.0001 ; ***p = 0.061; ****p = 0.0001.

Table 2. Analysis of microarray mRNA expression data for selected genes differentially expressed between 14 IUGR and 15 non-IUGR placentae with maternal vascular under-perfusion

Linear regression

Fold difference /j-value

IUGR/ adjusted

Affy ID Gene non-IUGR p-value for GA

205357_s_at AGTRI 0.56 0.0003 0,0002

208016 s at AGTRl 0.52 0.0005 0.0009

213183 s at CDKNIC 3.30 0.0138 0.0230

205630_at CRH 7.76 0.0002 0.0009

205629 s at CRH 8.00 0.0008 0.0031

208370 s at DSCRl 0.48 <0.0001 <0.0001

215253 s at DSCRl 0.51 O.0001 O.0001

216733 s at GATM 0.55 0.0003 0.001 1

203178_at GATM 0.59 0.0012 0.0041

206662 at GLRX 1.94 0.0001 0.0003

209276 s at GLRX 2.37 O.0001 O.0001

203913 s at HPGD 2.45 0.0002 0.0009

203914 x at HPGD 2.37 0.0006 0.0024

209541 at IGFl 0.48 O.0001 O.0002

209540 at IGFl 0.49 0.0003 0.001 1

202410 x at IGF2 0.39 0.0262 0.0252

210029 at INDO 0.57 O.0001 <0.0001

21051 1 s_at INHBA 5.66 0.0024 0.0079

207092_at LEP 6.23 0.0045 0.0101

210794_s_at MEG3 0.52 0.0002 0.0004

202016_at MEST 0.72 0.0008 0.0026

209803_s_at PHLDA2 1.27 0.0013 0.0017

209318 x at PLAGLl 0.62 0.0005 0.0007

207002 s at PLAGLl 0.72 0.0101 0.0073

208257_x_at PSG4 3.86 0.0057 0.0190

205602_x_at PSG4 5.10 0.0078 0.0196

Some genes are represented by more than one oligonucleotide probe set. The ^-values are from multiple linear regressions, testing for differences in mRNA for gestational age. Note: Since the statistical approach in generating the Supplementary table employed the Cross-Gene Error Model in GeneSpring, and the Benjamini-Hochberg correction for multiple comparisons, two genes shown here (IGF2, CDKNIC) do not appear in the Supplementary table. Fold

Difference IUGR vs. non Secreted Alfy lD Symbol Gβnbaπk IUGR T-tβst Description Affy ID Category 2 ? Function a disintegπn and metalloproteinase adipσgenesls, domain 12 (mettπn placental and

202952_s_a ADAM12 NM_00347 3.24 0.0007 alpha) 202952_s_at possible fetal growth a disintegπn and metalloproteinase adlpogenesis, domain 12 (me!lππ placental and

204943_at ADAM12 NM_02164 463 00043 alpha) 204943 at possible fetal growth a disintegπn and metalloproteinase domain 19 (meltπn

209765_at ADAM19 AF311317 1.86 00017 beta) 209765 at possible implantation assembly of extracellular

213974_at ADAMTSL3 AB033059 041 00008 KIAA1233 protein 213974_at possible matrices complement component 1, r

212067_S_a C1 R AL573058 051 0.0001 subcomponent 212067_s_ inflammation possible

CD44 antigen

(homing function and Indian blood

212063_at CD44 BE9O3880 060 0.0001 group system) 212063_at signaling possible

DKF2P586H2123 regeneration associated muscle

213661_at DKFZP586H21 AI671186 054 00007 protease 213661 at possible ectonucleoside triphosphate diphosphohydrolase nucleotide

209473_at ENTPD1 AV717590 202 00077 1 209473_at membrane p possible signalling placental protein 13- beta-gactoside

22O158_at LGALS14 NM_02012 5.10 O 0223 like protein 220158_at possible binding

213125_at OLFML2B AW007573 0.57 0.0002 olfaotomedin-like 2B 213125_at possible

HNOEL-iso protein, olfactomedin

218162_at OLFML3 NM_02019 0.34 00001 domain 218162 at possible receptor for protein C receptor, acivated protein

203650_at PROCR NM 00640 240 0.0018 endothelial (EPCR) 203650_at inflammation possible C cDNA clone

213362_at PTPRD N73931 041 O 0002 IMAGE 298260 213362_at membrane p possible protein tyrosine phosphatase,

214043_at PTPRD BF062299 047 O O0O2 receptor type, D 214043_at membrane p possible protein tyrosine regulates cell phosphatase, growth, mitosis,

200636_s. a PTPRF NM_00284 1 63 00028 receptor type, F 200636_s_ membrane p possible differentiation protein tyrosine phosphatase,

203038_at PTPRK NM 00264 059 0.0000 receptor type, K 203038_at membrane p possible

RelA-associated

21B849..S a RAI NM_00666 1 60 00042 inhibitor 218849_s_ , membrane p possible cell binding, cell signaling, and cytoskeletal

201287_s_a SDC1 NM_00299 490 00029 syndecan 1 201287_s_. signaling possible organization cell binding, cell

H sapiens signaling, and syndecan-1 gene cytoskeletal

201286_at SDC1 Z48199 666 00100 (exons 2-5) 201286_at signaling possible organization slit homolog 2 209897_s_a SLIT2 AF055585 0.46 00001 (Drosophila) 209897_s_at possible sushwepeat- contaimng protein,

204955_at SRPX NM_00630 047 00011 X-linked 204955_at possible sperm specific transmembrane

20250B at SSFA2 NM_00675 1 79 00026 antigen 2 202506_at possible signalling 2O415θIat STAB1 NMJM513 059 00004 stabilin 1 2O415θIat possible transports transferrin receptor transferrin and

208691_at TFRC BC001188 1 75 00038 (p90, CD71) 20869i_at transferπn tre possible iron thrombospondin, type I, domain

219477_s_a THSD1 NM_01867 054 o 0002 containing 1 219477_s_. cell adhesion possible

Thy-1 cell surface 208850_s_a THY1 AU558479 052 00020 antigen 208850_s_at possible

Thy-1 cell surface 208851_s_a THY1 AL161958 0.59 0.0052 antigen 20885i_s_at possible Fold

Difference IUGR vs. non Secreted

Affy ID Symbol Gonbank IUGR T-test Description Affy ID Category 2 ? Function

Ig superfamily

204787 at Z39IG NM_00726 058 00033 protein 204787_at possible

ALP, PLAP; alkaline phosphomonoester ase, function

204664 at ALPP NM_00163 7.82 00065 glycerophosphalase 204664_at yes unknown amine oxidase, copper containing 3 (vascular adhesion

204894_s_a AOC3 AF067406 051 00016 protein 1) 204894_s_. vascular adh yes bone morphogenetic

205431_s_a BMP5 NM_02107 051 00007 protein 5 205431_s_. TGFbeta farr yes glycoprotein hormones, alpha

204637. at CGA NM_00073 249 00018 polypeptide 204637_at βndocπne he yes collagen, type XIV, 212865..s_a COL14A1 BF449063 054 O 0009 alpha 1 (undulm) 212865 _s_at yes collagen, type XV, 203477. at COL15A1 NM_00185 060 00007 alpha 1 203477._at yes collagen, type XXI, 208096. _s_a COL21A1 NM_03082 059 O 0007 alpha 1 208096_s_at yes collagen, type V, 221729. at COL5A2 AL575735 052 00001 alpha 2 221729._at yes collagen, type V, 221730. at COL5A2 AL575735 0.54 00005 alpha 2 221730._at yes collagen, type V, 218975. at COL5A3 NM_01571 060 00001 alpha 3 218975._at yes collagen, type Vl, 212091..s_a COL6A1 AI141B03 0.49 00022 alpha 1 212091,_s_at yes collagen, type Vl, 213428. ,S_a COL6A1 AA292373 0.49 O 0000 alpha 1 213428._s_at yes collagen, type Vl, 201438 at COL6A3 NM_00436 048 O 0000 alpha 3 201438 at yes placental clock- corticotropin partuπtlon

205630_at CRH NM_00075 776 0,0008 releasing hormone 205630. at endocrine he yes trigger placental clock- corticotropin parturition 205629_s_a CRH BC0O2599 8.00 00022 releasing hormone 205629_s_. endocπne he yes trigger colony-stimulating angiogenesis; factor (CSF-1) trophoblastic 209716_at CSF1 M37435 0.59 00000 precursor 209716_at angiogeπesis yes differentiation defensin, alpha 1, myeloid-related 205033_s_a DEFA1 NM_00408 0 16 00151 sequence 205033_s_at yes ectonucleotide pyrophosphatase/p hosphodiesterase 2

210839_S_a ENPP2 D45421 0.41 00000 (autotaxin) 210839_s_ angiogenesis yes ectonucleotide pyrophosphatase/p hosphodiesterase 2

209392_at ENPP2 L35594 055 00001 (autotaxin) 209392_at angiogenesis yes mediates platelet 202994. s_a FBLN 1 295331 1 97 0.0029 flbulin 1 202994 s at yes adhesion mediates platelet

207835 at FBLN1 NM 00648 2.82 00113 fibulin i 207835_at yes adhesion 202709lat FMOD NM_00202 0.59 00007 fibromodulin 202709_at TGFbeta pat yes promotes fetal survival by suppressing maternally derived growth proinflammatory differentiation factor cytokines w/ιn

221577 ,x_a GDF15 BC000529 431 00169 15 221577_x_, TGFbeta farr yes uterus placental growth control, trophoblast invasion,

211508_s_a GH2 AF006060 5.78 0.0304 growth hormone 2 211508_s endocrine he yes somatogeπesis hepatocyte growth factor (hepapoletin 209961_s_a HGF M60718 055 00029 A, scatter factor) 209961_s_ growth factoi yes insulin-like growth factor 1 209541_at IGF1 AI97249S 0.48 0.0001 (somatomedin C) 209541_at growth factor yes Fold

I Difference

IUGR vs. non Secreted

Affy ID Symbol Genbank IUGR T-test Description Affy ID Category 2 ? Function insulln-llke growth factor 1

209540_at IGF1 M29644 049 00003 (somatomedin C) 209540_at growth factoi yes inhibin, beta A

(activln A, activin

AB alpha supports

210511. s _a INHBA M13436 5.66 00045 polypeptide) 210511_s_at yes somatic growth lamlnln, alpha 2

(merosin, congenital basement muscular membrane

213519_8_a LAMA2 AI078169 040 O O00O dystrophy) 213519_s_at yes component laminin, alpha 2

(merosin, congenital basement muscular membrane

205116_at LAMA2 NM_00042 043 0.0000 dystrophy) 205116_at yes component laminin, alpha 2

(merosin, congenital basement muscular membrane

216840_S_a LAMA2 AK026329 047 00000 dystrophy) 216840_s_at yes component matrix

219407_s_a LAMC3 NM_00605 052 00005 laminin, gamma 3 219407_s_at yes glycoprotein homeostasis 2) placental development 3) leptin (obesity may play role in

207092_at LEP NM_00023 623 00089 homolog, mouse) 207092_at hormone yes angiogenesis lipoprotein/ cholesterol metabolixm, particularly endothelial

219181_at LIPG NM_O0603 049 0.0003 lipase, endothelial 219181_at yes (vascular) connective tissue

202998_s_a L0XL2 AF117949 051 00001 lysyl oxidase-like 2 202998_s_. collagen mat yes biogenesis

Microfibπl- extracellular associated matrix

209758_s_a MFAP5 U37283 1 97 00003 glycoproteιn-2 209758_s_at yes glycoprotein matrix extracellular metalloproteinase 2 matπx

201069_at MMP2 NM 00453 059 00010 (gelatinase A) 201069_at yes remodelling basement membrane

202008_s_a NID1 NM_00250 0.60 00001 nidogen (enactin) 202008_s_at yes component basement membrane

202007_at NID1 8F940043 060 00002 nidogen (enactin) 202007_at yes component matrix metalloproteinas e, produced by pregnancy- trophpblast, iGF associated plasma binding proteιn4

201981_at PAPPA AA148534 379 00052 protein A 201981_at yes protease matπx metalloproteinas e, produced by pregnancy- trophpblast, IGF associated plasma binding proteιn4

201982. s. a PAPPA NM.00258 4.95 0.0126 protein A 201982_S_at yes protease procollagen C- endopeptldase

202465_at PCOLCE NM_00259 045 00028 enhancer 202465_at yes

PAPPA2 gene; mstalloendopept

211918_x_a PLAC3 AF311940 3 11 0.0128 placenta-specific 3 211918_x_at yes idase activity

PAPPA2 gene, melalloeπdopept

213332_at PLAC3 AL031290 318 00028 placenta-specific 3 213332_at yes idase activity protease, seπne, 11 inhibits TGF-

201185_at PRSS11 NM_00277 2.78 00063 (IGF binding) 201185_at yes beta signalling immune pregnancy specific modulation; beta-1-glycoproteιn enhances

210195_s_a PSG1 M34715 326 00450 1 210195_s_at yes embryo growth immune pregnancy specific modulation, beta-1-glycoproteιn enhances

210196_s_a PSG1 M33663 3.29 0.0500 1 210196_s_at yes embryo growth Fold

Difference

IUGR vs. non Secreted

Affy ID Symbol Gβnbank IUGR T-tθst Description Affy ID Category 2 ? Function immune pregnancy specific modulation; beta-1-glycoproteιn enhances

208106_x_a PSG1 NM. 00278 4.12 00061 6 20810S_x_at yes embryo growth pregnancy specific enhances bθta-1-glycoproteιn embryo growth 20657O_S_a PSG3 NM_00278 306 00273 11 206570_s_at yes (mouse models) pregnancy specific enhances beta-1-glycoproteιn embryo growth 215821_x_a PSG3 R32065 342 00037 3 215821_x_at yes (mouse models) pregnancy specific enhances beta-1-g]ycoprotein embryo growth 208134_x_a PSG3 NM_03124 371 00050 2 208134_X_at yes (mouse models) pregnancy specific enhances beta-1-glycoprotein embryo growth 203399_x_a PSG3 NM_02101 377 00050 3 203399_x_at yes (mouse models) pregnancy specific enhances beta-1-glycoproteιn embryo growth 211741_x_a PSG3 BC005924 381 00056 3 211741_x_at yes (mouse models) pregnancy specific enhances beta-1-glycoproteιn embryo growth

204830. x_ a PSG3 NM_00278 3.90 0.0069 4 204830_x_at yes (mouse models) pregnancy specific beta-1-glycoprotθln 208191_x_a PSG4 NM_00278 3.77 00084 4 208191_x_at yes unclear pregnancy specific beta-1-glycoproteιn 208257_x_a PSG4 NM_00690 3.86 00089 1 208257_x_at yes unclear pregnancy specific beta-1-glycoprotein 205602_x_a PSG4 NM_00278 5 10 00155 7 205602_x_at yes unclear pregnancy specific beta-1-glycoproteιn 207733_x_a PSG9 NM_00278 324 0.0080 9 207733_x_at yes Unclear pregnancy specific beta-1-glycoproteιn 209594_x_a PSG9 M34421 3.48 00086 9 209594_x_at yes unclear pregnancy specific bela-1-glycoprotein 209738_x_a PSG9 M31125 3.92 00081 6 209738_X_at yes unclear pregnancy specific beta-1 -glycoprotein 210126_at PSG9 M94890 441 00156 9 210126_at yes unclear spondin 1, extracellular matrix

20943B_at SPON1 AB018305 0.40 0.0000 protein 209436_at angiogenesi: yes tissue factor placental 210665_at TFPI AF021834 346 00090 pathway inhibitor 210665_at extracellular yes hemostasis tissue factor tissue 209277_at TFPI2 AL574096 1 76 00017 pathway inhibitor 2 209277_at extracellular yes remodelling tissue inhibitor of angiogenesis

203167_at TIMP2 NM_00325 380 00071 metalloproteinase 2 203167_at yes inhibitor bone 205807_s_a TUFT1 NM_02012 1.66 00013 tuftelin 1 205807_s extracellular yes mineralization

WAP four-disulfide 219478_at WFDC1 NM_02119 0.54 00025 core domain 1 219478_at extracellular yes wingless-type

MMTV Integration site family member

205648_at WNT2 NM_00339 0.52 00011 2 205648_at wnt ligand yes cDNA clone 44702_at 7r>3 R77097 1.89 00022 IMAGE 144061 44702_at

ATP-blnding cassette, sub-family

G (WHITE),

204567 , S_aABCG1 NM_00491 1 83 00016 member 1 204567_s_. cholesterol transport abhydrolase 218739_at ABHD5 NM_01600 1 90 00020 domain containing 5 218739_at acyl-CoA synthetase long- chain family

202422_s_a ACSL4 NM_02297 271 0.0047 member 4 202422 s at unclear role in blood pressure 201752_s aADD3 AI763123 0.60 0.0002 adducin 3 (gamma) 201752_s_. blood pressure regulation Fold Difference IUGR vs. non Secreted

Affy ID Symbol Genbank IUGR T-tast Description Affy ID Category 2 ? Function adipose lipid diffβrentiallon- accumulation/

209122_at ADFP BC005127 1 92 00012 related protein 209122_at adiopogenesis 204120_S_a ADK NM_00112 1 66 00003 adenosine kinase 204120_s_ signaling angiotensin Il

208016_S_a AGTR1 NM_00483 052 0.0005 receptor, type 1 208016_S_. blood pressure angiotensin Il

205357_s_a AGTR1 NM_O00B8 0.56 O 0006 receptor, type 1 205357_s_. blood pressure adenosylmethionine

201197_at AMD1 M21154 1 60 00001 decarboxylase 1 201197_at adenosylmethionine

201196_S a AMD1 M21154 1.67 O 0003 decarboxylase 1 201196_s_at breast cancer anti- estrogen resistance

204032_at BCAR3 NM_00356 273 00044 3 204032 at

B-cell

CLL/lymphoma 6

{zinc finger protein

203140_at BCU6 NM_00170 206 0.0071 51) 203140_at basic leucine zipper

217809_at BZW2 NM.01403 1 94 0,0006 and W2 domains 2 217809_at chromosome 14 open reading frame

219316_s a C14orf58 NM_01779 1 62 00036 58 219316 s_ SLC family 215411_s_a C6orf4 AL008730 1 73 00045 215411_s_. TRAF3IP2

CAP, adenylate cyclase-aεsociated

212554 at CAP2 N90755 265 0.0062 protein, 2 (yeast) 212554_at 202965_s_a CAPN6 NM_01428 204 0.0019 calpain 6 202965_s_at 202966_at CAPN6 NM_01428 266 00041 calpain 6 202966_at core-binding factor, runt domain, alpha

205529_s_a CBFA2T1 NM_00434 048 00026 subuπrt 2 205529_s_at cDNA clone

212501_at CEBPB AL564683 1 65 0.0007 CSODM007YK12 212501_at chromosome

204759_at CHC1L NM 00126 0.49 0.0000 condensation 1-like 204759_at 212624_s a CHN1 BF339445 047 00002 chaemeπn-1 212624_s_at clattiπn, light

211043 3_a CLTB BC006332 1 60 0 0026 polypeptide (Lcb) 211043_s_at 205518_s_a CMAH NM_00357 1 83 00008 205518_s_ glycosylation 201445_at CNN3 NM 00183 056 0.0003 calpoπin 3, acidic 201445_at cytoskeletal regulation 203642_S_a C0BLL1 NM_01490 2 18 0.0032 COBL-like 1 203642_s_at cysteine-πch protein

205081_at CRIP1 NM_00131 1.86 0.0098 2 205081_at colony stimulating factor 2 receptor,

205159_at CSF2RB AV756141 229 0.0018 beta, low-affinity 205159_at cytochrome b5

202263 at CYB5R1 NM 01624 1.89 00029 reductase 1 (B5R 1) 202263_at mitochondrial function cytochrome b-245, beta polypeptide

(chronic granulomatous

203923 s_a CYBB AI308863 0.43 0.0005 disease) 203923_s_. mitochondrial function cytochrome b-245, beta polypeptide

(chronic granulomatous

203922_s_a CYBB AI308863 045 00007 disease) 203922. s_ mitochondrial function cytochrome P450, family 19, subfamily cholesterol

203475_at CYP19A1 NM_00010 3.77 0.0063 A, polypeptide 1 203475_at synthesis

DKFZP564O123

CHMP2B chromatin modifying

202537_s_a DKFZP564O12 NM_01404 2 16 00068 protein 2B 202537_s_at downregulated in 204135_at D0C1 NM_01489 052 0.0014 ovaπan cancer 1 204135_at ATPase dihydropyπmidinase-

201431_s_a DPYSL3 NM_00138 051 00007 like 3 201431_s_at

Down syndrome angiogenesis/ critical region gene endoethelial

208370 s a DSCRI NM_00441 048 00001 1 208370 _s_. angiogenesis proliferation Fold

I Jifference

IUGR vs. non Secreted

Affy ID Symbol Genbank IUGR T-test Description Affy ID Category 2 ? Function

Down syndrome angiogenesis/ critical region gene endoelhelial

21S253_s_a DSCR1 AL049369 051 OO001 1 215253_s_. angiogenesis proliferation

Epstein-Barr virus

205419_at EBI2 NM_00495 0.49 00002 induced gens 2 205419_at membrane receptor cell mediated

Epstein-Barr virus immune

219424_at EBI3 NM_O0575 438 O 0013 induced gene 3 219424_at cytoskelβtal regulation responses

EF hand domain

209343_ai EFHD1 BC002449 264 00021 containing 1 209343_at calcium binding edonucleotide pyrophosphatase/p

205066_S_a ENPP1 NM_00620 052 00055 hosphodiesterase 1 205066. s_ metabolic enzyme erythrocyte extracellular membrane protein matrix

201718_S_a EPB41L2 BF511685 060 00002 band 4 1-IiKe 2 201718_s_at component

Fc fragment of IgE, high affinity I, receptor for,

2Q4232_al FCER1G NM_00410 060 O.0007 gamma polypeptide 204232_at Inflammation

Fc fragment of IgG, high affinity Ia,

214511_x_a FCGR1A L03419 056 00013 receptor for (CD64) 214511_x_. Inflammation

203647 S a FDX1 NM_00410 261 00042 ferredoxin 1 203647_s_. steroid metabolism

203646_at FDX1 NM_0D410 363 0.0107 ferredoxin 1 203646_at steroid metabolism hypothetical protein

FLJ10116; DSU

219648_at FU10116 NM_01800 1.67 0.0050 protein 219648_at hypothetical protein

218614_at FU10S52 NM_01816 060 00005 FU10652 218614_at multiple CZ- domains with two transmembrane

220603_s_a FU11175 NM 01834 060 O 0019 regions 2 220603_S_at

218546_at FLJ14146 NM_02470 1 66 00014 C1orf115 218546_at multiple C2- domams with two transmembrane

22O122_at FLJ22344 NM_02471 0.43 00025 regions 1 220122_at colon carcinoma related protein, vestigial-related

220327 at FU38507 NM 01620 277 00024 protein 220327_at

206015_s_a FOXJ3 NM_01494 1 64 O0014 forkhead box J3 206015_S_at fused toes homolog

218373_at FTS NM_02247 1 70 00058 (mouse) 218373_at

GABA(A) receptor- associated protein

208868_S_a GABARAPL1 BF125756 1 66 0.0036 like 1 208868_s_. subcellular trafficking?

GATA binding

209603 at GATA3 AI796169 2.04 O.00O5 protein 3 209603_at glycine aminotransferase

(L-argιnine:glycine

216733_s_a GATM X86401 055 00002 aminotransferase) 216733_s_. imprinted gene glutaredoxin

206662_at GLRX NM_00206 1 94 00002 (thioltransferase) 206662_at glutaredoxin

209276_s_a GLRX AF162769 2.37 0.0001 (thioltransferase) 209276_s_at

G protein-coupled

221814_at GPR124 BF511315 057 00001 receptor 124 221814_at membrane receptor guanylate cyclase

221942_s_a GUCY1A3 AI719730 0.45 0.0000 1, SOlUbIe, alpha 3 221942_s_at guanylate cyclase

211555_s_a GUCY1B3 AF020340 048 0.0000 1, soluble, beta 3 211555_s_at guanylate cyclase

203817_at GUCY1B3 W93728 053 00001 1, soluble, beta 3 203817_at

GULP, engulfment adaptor PTB

204237_at GULP1 NM_01631 245 00054 domain containing 1 204237_at hexosaminidase B

201944_at HEXB NM_00052 1 67 00005 (beta polypeptide) 201944_at

Homo sapiens histone i, H1c, mRNA (cDNA clone

209398 at HIST1H1C BC002649 3.14 0.0005 IMAGE:3608862). 209398_at histone Fold Difference IUGR vs. non Secreted

Affy ID Symbol Genbank IUGR T-test Description Affy ID Category 2 ? Function high-mobility group

203744_at HMGB3 NM_00534 1 73 0.0009 box 3 203744_at 216548_x_a HMGB3 AL049709 245 000O2 216548lx_at homeodomain-only

211597_s_a HOP AB059408 356 00063 protein 211597_s_at hydroxyprostaglandi n dehydrogenase

203914_x_a HPGD NM_00086 237 00009 15-(NAD) 203914_x_. prostaglandin synthesis hydroxyprostaglandi n dehydrogenase

203913_s_a HPGD NM_0008B 245 00003 15-(NAD) 203913_s_ prostaglandin synthesis hydroxyprostaglandi n dehydrogenase

211548_s_a HPGD J05594 257 0.0017 15-(NAD) 211548_s_. prostaglandin synthesis hydroxyprostaglandi n dehydrogenase

211549_s_a HPGD US3296 3.29 00007 15-(NAD) 211549_s_ prostaglandin synthesis hydroxy-delta-5- steroid

204515_at HSD3B1 NM_00086 279 00024 dehydrogenase 204515_at steroid metabolism heat shock 27kDa

201841 B a HSPBI NM 00154 1 92 00038 protein 1 201841_s_. metabolic stress protection?

HIV-1 Tat interactive protein 2,

209448_at HTATIP2 BC002439 1 99 00023 3OkDa 209448_at intercellular adhesion molecule

204683_at 1CAM2 NMJJ0087 O 57 0.0008 2 204683_at immune system intestinal epithelial cell intestinal cell (MAK- proliferation and

204569_at ICK NM_01492 4.17 00143 like) kinase 204569_at differentiation iduronate 2- suifatase (Hunter

212223_at IDS AV703259 1 79 00035 syndrome) 212223_at iduronate 2- sulfatase (Hunter

206342_x_a IDS NM .00612 1.94 00071 syndrome) 206342_x_at iduronate 2- sulfatase (Hunter

202439.s. a IDS NM_00020 253 00038 syndrome) 202439_S_ϊ immediate early

218611_at IER5 NM_01654 1 76 00007 response 5 218611_at

Interferon, gamma-

208965_s_ .a IFH 6 BG256677 058 0.0004 inducible protein 16 208965_s_ transcription factor interferon, gamma-

208966 X a IFH 6 AF208043 059 0.0001 inducible protein 16 208966_x_. transcription factor interferon, gamma-

206332_s_a IFI16 NM_00553 0.60 00002 lnducibls protein 16 206332_s_ . transcription factor immunoglobulin superfamily,

202421_at IGSF3 AB007935 2 16 00019 member 3 202421_at frneriBUMii i receptor accessory interleukin

205227_at IL1RAP NM.00218 1.65 00026 protein 205227_at signaling signaling interleukin 1 receptor accessory interleukin

210233_at IL1RAP AF167343 3.52 0.0021 protein 210233_at signaling signaling indoleamme-pyrrole - tolerance of fetal

210029_at INDO M34455 0.57 00002 2,3 dioxygenase 210029_at immune system hemiallograft? insulin Induced

201626_at INSIG1 BG292233 1 83 00052 gene i 201626_at intracellular trafficking insulin induced

201627_s_a INSIG1 BE300521 1 88 0.0055 gene i 201627_s_ intracellular trafficking

214660_at ITGA1 X68742 052 00010 integrin, alpha 1 214660_at trophoblast invasion potassium channel, subfamily K,

205952_at KCNK3 NM_ 00224 042 00089 member 3 205952_at potassium voltage- gated channel, delayed-redifier,

205968 at KCNS3 NM_00225 053 0.0006 subfamily S 205968 at Fold

Difference

IUGR vs. πon Socrotβd I

Affy lD Symbol Genbank IUGR T-test Description Affy ID Category 2 ? Function microfibπllar- assoαated protein 3

205442_al KIAA0626 NM_02164 379 00027 like 205442_at call adhesion unknown

212327_at KIAA1102 AK026815 342 00021 LIM domain 212327_at unknown KIAA1102 protein,

212328_at KIAA1102 AB029025 4 10 00024 LIM domain 212328_at unknown

KIAA1102 protein,

212325_at KIAA1102 AK026815 447 00030 LIM domain 212325 at

213085_s_a KIBRA AB020676 1 98 0.0036 KIBRA protein 213085ls_at klnesin heavy chain

203087_s_a KIF2 NM_00452 2.55 00029 member 2 203087_s_at

205978_ai KL NM_00479 0.44 00020 Klotho 205978_at vitamin D metabolism lysosomal associated protein transmembrane 4 integral to

214039_s_a LAPTM4B T15777 0.59 0.0003 beta 214039_s_at membrane lymphoid enhancer-

221558_s_aLEF1 AF288571 0.56 0.0030 binding factor 1 221558_s_at nuclear protein

LIM domain kinase cytoskeltal

202193_at UMK2 NM_00556 1 82 0.0021 2 202193_at organization signallin, ^* lymphocyte adaptor stimulates

203320_at LNK NM_00547 0.59 00006 protein 203320_at intracellular adaptor prot hematopoiesis

MRNA, cDNA

DKFZpS66E183

(from clone

213248_at LOC221362 AL577024 0.38 O.0O13 DKFZp566E183) 213248_at unclear cDNA clone nucleic acid

214751_at LOC90333 BE541042 2.32 00016 IMAGE 3450599 214751_at binding cell adhesion, signal

206953_s_a LPHN2 NM_01230 0.51 0.0020 latraphilin 2 206953_s_. G-proteiπ coupled recep transduction leucine rich repeat

218816_at LRRC1 NM_01821 264 0.0026 containing 1 218816_at unkown tight junction

212251_at LYRIC AI972475 057 00004 LYRIC/3D3 212251_at maturation v-maf musculoaponeurotic fibrosarcoma oncogene homolog cellular stress

36711_at MAFF AL021977 261 0.0011 F 36711_at stress response reponse v-maf musculoaponeurotic fibrosarcoma oncogene homolog cellular stress

205193_at MAFF NM_01232 344 00026 F 205193_at stress response reponse calicum ion mannosidase, binding, alpha, class 1C, carbohydrate

214180_at MAN1C1 AW340588 273 O 0014 member 1 214180_at golgi enzyme metabolism mannosidase, binding, alpha, class 1C, carbohydrate

218918_at MAN1C1 NM_02037 368 00041 member 1 218918_at golgi enzyme metabolism transcription enhancer factor 2, myogenic

209200_at MEF2C AL536517 0.60 00008 polypeptide C 209200_at differentiation maternally growth

210794__{S -}a MEG3 AF119863 052 0D001 expressed 3 210794_s_. impnnted gene suppressor MeIsI, myeloid ecotroplc viral integration site 1 homeobox

204069_at MEIS1 NM 00239 051 00001 homolog (mouse) 204069_at protein MOB1 MDS One

Binder kinase mitotic activator-like 2B checkpoint

219265_at MOBKL2B NM_02476 1 72 00054 (yeast) 219265_at regulation cytoskeltal

201976_s_a MYO10 NM_01233 0.59 0.0002 myosin X 201976_s_at organization

212365_at MYO1B BF215996 057 00001 myosin IB 212365_at cross links actin nuclear receptor subfamily 3

(glucocorticoid glucocorticoid

216321 s a NR3C1 X03348 059 00003 receptor) 216321_s_. endocrine signaling receptor gene

203675_at NUCB2 NM_00501 1 86 0.0041 nucleobindin 2 203675_at calcium binding calcium binding ornithine decarboxylase

201772 at OAZIN NM_01587 1.75 0.0042 antizyme inhibitor 201772 at polyamiπe synthesis Fold

Difference

IUGR vs. non Secreted

AfTy ID Symbol Gonbank IUGR T-test Description Affy ID Category 2 ? Function transmembrane emp24 domain

208837_at P24B BC000027 1 60 00025 containing 3 208837_at intracellular trafficking domain 2

219165_at PDLIM2 NM .02163 230 00018 (mystique) 219165_at

PDZ domain containing RING

212915_at PDZRN3 AL569804 052 00038 finger 3 212915_at ubiqurtin ligase progesterone receptor membrane

201701_s_a PGRMC2 NM_00632 1 60 00014 component 2 201701_s_. membrane protein phospholipase C-

213309_at PLCL2 AL117515 2 15 00021 like 2 213309_at peripheral myelin

210139_s_a PMP22 L03203 0.59 00000 protein 22 210139_s_at protein kinase, AMP activated, gamma 2 non-catalytic

218292_s_a PRKAG2 AF087875 1 91 00005 subunrt 218292_s_ stress sensor'

RAS guanyl releasing protein 1 ras family,

(calcium and DAG- membrane

205590 at RASGRP1 NM_00573 5.88 00168 regulated) 205590 at signalling

203224j3t RFK BF340123 1 75 00012 riboflavin kinase 203224_at

203225_S_a RFK NM_01833 1.78 O0048 riboflavin kinase 2O3225_s_at

S100 calcium

204351_at S100P NM_00598 2.10 00025 binding protein P 204351_at spermidine/spermin e N1-

210S92_S_ .a SAT M55580 1 68 O.0006 acety (transferase 210592_S_at spermidine/spermin e N1-

213988_s_ a SAT BE971383 1.91 O.0007 acetyltransferase 213988_s_at sialyltransferase 10

(alpha-2,3-

213355_at S1AT10 AI989567 205 0.0006 sialyltransferase Vl) 213355_at glycosylalion sialyltransferase 10

(alpha-2,3-

210942 s a SIAT10 AB022918 2.11 0.0008 sialvltransferase Vl) 210942_s _. glycosylation solute earner family 11 (proton-coupled divalent metal ion placental iron

203124_s_a SLC11A2 AF046997 1 63 0.0036 transporters) 203124. s metal ion transport transer solute carrier family 16 (monocarboxylic acid transporters), lactic acid

202856_s_a SLC16A3 NM__00420 222 00037 member 3 202856_s_ monocarboxylalβ transp transporters cDNA clone zinc membrane 202088_at SLC39A6 AI635449 1 91 00033 IMAGE 2233416 202088_at metal ion transport transporter solute carrier family

39 (zinc transporter),

209267_S_a SLC39A8 AB040120 3.52 00059 member 8 2O9267._s_. metal Ion transport solute earner family

39 (zinc transporter), 219869_s_a SLC39A8 AW139759 393 00054 member 8 219869_s. metal ion transport solute earner family 7 (cationic amino acid transporter, y+

212295_s_a SLC7A1 AA148507 302 00128 system) 212295_s_. catιonιcamιno acιd traniAA transporter solute carrier family

7 (cationic amino add transporter, y+

201195. s_a SLC7A5 AB018009 275 0.0066 system) 201195_S_ cationic amino acid transport solute earner organic anion transporter family, 204368_at SLCO2A1 NM_00563 245 00006 member 2A1 204368 at prostaglandin transport

SWI/SNF related, matrix associated, regulator of

212167_s_a SMARCB1 AK021419 1 63 00031 chromatin 212i67_s_at snail nomolog 2 213139_at SNAI2 AI572079 0.51 0.0035 (Drosophila) 213139_at Fold

Difference IUGR vs. non Secreted

Affy ID Symbol Genbank IUGR T-test Description Affy ID Category 2 ? Function 218404_at SNX10 NM_01332 277 0.0020 sorting nexin 10 218404 at gaπglioside-induced differentiation- associated protein 1

212558_at SPRY1 BF50S662 055 00002 like 1 212558_at seπne/threonine kinase 3 (STE20

204068_at STK3 NM_00628 1.89 00021 homolog. yeast) 204068_at apoptosis? transcription factor

222146_s_aTCF4 AK026674 053 00001 4; SEF2B 222146_s_at transcription factor

204653_at TFAP2A BF343007 1.83 00021 AP-2 alpha 204653_at transforming growth factor beta 1

209651_at TGF81I1 BC001830 058 0.0004 induced transcript 1 209651_at hypothetical protein

219975 x a THEDCI NM_01832 880 0.0155 FLJ11106 219975_x_.thιolesterase tight junction protein

2 (zona occludens

202085 at TJP2 NM_00481 1 98 00024 2) 202085_at tankyrase, TRF1- interacting ankyπn- related ADP-πbose

218228_s_a TNKS2 BF060683 1.60 0.0043 polymerase 2 218228_s_at zinc finger, CCHC

2190B2_s_a ZCCHC2 BE676543 1 66 00002 domain containing 2 219062_s_at zinc finger 203603_S_a ZFHX1B NM_01479 053 0.0002 homeobox 1b 203603_s_at

Claims

WE CLAIM:

1. A method of diagnosing intrauterine growth restriction comprising (a) measuring the level of gene expression of a target intrauterine growth restriction related gene in a subject sample; (b) comparing the level of gene expression of the target gene in the subject sample with intrauterine growth restriction to the level of gene expression of the target gene in one or more healthy subject sample(s); wherein a statistically significantdecrease or increase in gene expression of the target gene in the subject sample with intrauterine growth restriction when compared to the gene expression of the target gene in the healthy subject sample indicates a diagnosis of intrauterine growth restriction.

2. The method of claim 1, wherein the subject sample is derived from the mother or the fetus.

3. The method of claim 2, wherein the subject sample comprises a tissue or blood sample selected from the group comprising placental tissue, amniotic fluid, and blood.

4. The method of claim 1, wherein measuring the level of gene expression is performed by Northern blot.

5. The method of claim 1 , wherein measuring the level of gene expression is performed by DNA chip.

6. The method of claim 1 , wherein the subject sample is derived from a person at risk of intrauterine growth restriction.

7. The method of claim 6, wherein the person at risk of intrauterine growth restriction exhibits a condition selected from the group consisting of poor nutrition, heart disease, high blood pressure, smoking, drug abuse, alcohol abuse, viral or bacterial infections, congenital or chromosomal abnormalities, maternal medical conditions such as sickle cell anemia or lupus, multiple gestations, and exposure to environmental toxins.

8. The method of claim 1, wherein the intrauterine growth restriction related gene is selected from the group consisting of CD81, CDKNlC, DCN₅ DIO3, DLKl, GATM, GNAS, GRBlO, HYMAI, IGF2, IGF2R, MEG3, MEST, MKRNl, NDN₅ NNAT, MESTO, PEG3, PLAGLl, PON2, PPPlCC, SGCE, SNRPN, SNURF, PHLDA2, UBE3A, and ZIM2.

9. The method of claim 8, wherein the intrauterine growth restriction related gene is PHLDA2.

10. The method of claim 8, wherein the intrauterine growth restriction related gene is MEST.

11. The method of claim 1 , wherein the level of gene expression of the target gene in one or more healthy subject sample(s) is represented by a normalized gene expression value.

12. A method of diagnosing intrauterine growth restriction comprising (a) measuring the level of gene expression of a target maternally expressed/paternally repressed imprinted gene and the level of expression of a target maternally repressed/paternally expressed imprinted gene in a subject sample; (b) calculating a first ratio of gene expression of the maternally expressed/paternally repressed imprinted gene to the maternally repressed/paternally expressed imprinted gene in the subject sample;

(c) comparing the first ratio of gene expression from the subject sample to a ratio of gene expression from one or more healthy subject sample(s); wherein a statistically significantdecrease or increase of the ratio from the subject sample when compared to the ratio from the healthy subject sample indicates a diagnosis of intrauterine growth restriction.

13. The method of claim 12, wherein the subject sample is derived from the mother or the fetus.

14. The method of claim 13, wherein the subject sample comprises a tissue or blood sample selected from the group comprising placental tissue, amniotic fluid, and blood.

15. The method of claim 12, wherein measuring the level of gene expression is performed by Northern blot.

16. The method of claim 12, wherein measuring the level of gene expression is performed by DNA chip.

17. The method of claim 12, wherein the subject sample is derived from a person at risk of intrauterine growth restriction.

18. The method of claim 17, wherein the person at risk of intrauterine growth restriction exhibits a condition selected from the group consisting of poor nutrition, heart disease, high blood pressure, smoking, drug abuse, alcohol abuse, viral or bacterial infections, congenital or chromosomal abnormalities, maternal medical conditions such as sickle cell anemia or lupus, multiple gestations, and exposure to environmental toxins.

19. The method of claim 12, wherein the maternally expressed/paternally repressed imprinted gene is PHLDA2.

20. The method of claim 12, wherein the maternally repressed/paternally expressed imprinted gene is MEST.

21. The method of claim 12, wherein the ratio of gene expression of the target genes in one or more healthy subject sample(s) is represented by a normalized ratio of gene expression value.

22. A method of identifying genes relating to intrauterine growth restriction comprising (a) measuring the level of gene expression of a target gene in one or more subject sample(s) diagnosed with intrauterine growth restriction;

(b) measuring the level of gene expression of the target gene in one or more healthy subject sample(s) not suffering from intrauterine growth restriction;

(c) comparing the gene expression of the target gene in the subject sample with intrauterine growth restriction to the gene expression of the target gene in the healthy subject sample; wherein a statistically significantdecrease or increase in gene expression of the target gene in the subject sample with intrauterine growth restriction when compared to the gene expression of the target gene in the healthy subject sample indicates a gene relating to intrauterine growth restriction.

23. The method of claim 22, wherein the subject sample is derived from the mother or the fetus.

24. The method of claim 23, wherein the subject sample comprises a tissue or blood sample selected from the group comprising placental tissue, amniotic fluid, and blood.

25. The method of claim 22, wherein measuring the level of gene expression is performed by Northern blot.

26. The method of claim 22, wherein measuring the level of gene expression is performed by DNA chip.

27. A kit for diagnosing intrauterine growth restriction comprising: (1) one or more oligonucleotide probe(s) directed to one or more intrauterine growth restriction related gene(s); and (2) a control oligonucleotide probe directed to a gene that is not related to intrauterine growth restriction.

28. The kit of claim 27 wherein the reagents and equipment for purifying nucleic acids from tissue or fluid samples includes oligonucleotide probes.

29. The kit of claim 27 wherein the oligonucleotide probes comprise sequences complementary to portions of the genes selected from the group consisting of CD81 , CDKNlC, DCN, DIO3, DLKl, GATM, GNAS, GRBlO, HYMAI₅ IGF2, IGF2R, MEG3, MEST, MKRNl, NDN, NNAT, MESTO, PEG3, PLAGLl, PON2, PPPl CC, SGCE, SNRPN, SNURF, PHLD A2, UBE3 A, and ZIM2.