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WO2007097741A1 - Méthode de diagnostic d'une restriction de croissance intra-utérine - Google Patents

Méthode de diagnostic d'une restriction de croissance intra-utérine Download PDF

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WO2007097741A1
WO2007097741A1 PCT/US2006/005632 US2006005632W WO2007097741A1 WO 2007097741 A1 WO2007097741 A1 WO 2007097741A1 US 2006005632 W US2006005632 W US 2006005632W WO 2007097741 A1 WO2007097741 A1 WO 2007097741A1
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gene
iugr
gene expression
subject sample
growth restriction
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Benjamin Tycko
Harshwardhan M. Thaker
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Columbia University in the City of New York
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Columbia University in the City of New York
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • 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.
  • IUGR intrauterine growth restriction
  • IUGR intrauterine growth restriction
  • 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.
  • IGF2 receptor IGF2r
  • IGF2r insulin-like growth factor 2 receptor
  • the paternal IGF2 allele is expressed and the maternal IGF2 allele is repressed
  • 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.
  • 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).
  • UPDs rare uniparental chromosomal disomies
  • IUGR is typically defined as a fetus or newborn infant whose weight is below the 10th percentile for its gestational age.
  • 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.
  • 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.
  • 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.
  • 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.
  • IUGR intrauterine growth restriction
  • 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.
  • IUGR-related or IUGR-associated genes are referred to herein as IUGR-related or IUGR-associated genes.
  • determination of elevated or reduced expression levels of IUGR-related genes in maternal or fetal samples can be a reliable predictor of IUGR.
  • 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.
  • 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 im
  • 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.
  • 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, IGFl 5 INDO, INHBA, LEP, PSG4 or a functionally equivalent gene.
  • an IUGR-associated gene selected from the group consisting of CD81, CDKNlC, DCN, DIO3, DLKl, GATM, GNAS, GRBlO, HYMAI, IGF2, IGF2R, MEG3, MEST, MK
  • 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.
  • the IUGR related gene measured is PHLD A2 and/or MEST, or functionally equivalent genes.
  • 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.
  • 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.
  • cDNA can refer to a single-stranded or double-stranded DNA molecule.
  • DNA strand is complementary to the messenger RNA ("mRNA") transcribed from a gene.
  • mRNA messenger RNA
  • a double-stranded cDNA molecule one DNA strand is complementary to the mRNA and the other is complementary to the first DNA strand.
  • 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.
  • 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.
  • 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.
  • 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.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • two nucleic acid molecules are "functionally equivalent" when they share two or more quantifiable biological functions.
  • 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.
  • 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.
  • 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
  • a stringent hybridization washing solution may be comprised of 40 mM NaPO 4 , pH 7.2, 1-2% SDS and 1 mM EDTA.
  • 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).
  • sequence refers to a nucleic acid molecule having a particular arrangement of nucleotides, or a particular function, e.g. a termination sequence.
  • the term "subject" refers to an animal, e.g. , a mammal.
  • the subject is a human.
  • the subject is a human fetus or a newborn infant.
  • the subject is a pregnant human female.
  • the term “derived” means "obtained from,” “descending from,” or “produced by.”
  • the term derived refers to obtaining the tissue or fluid samples from the 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.
  • the term derived refers to the sampling of multiple subjects to obtain a normalized, average value.
  • 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.
  • 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.
  • 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
  • fluorescent compounds e.g., fluorescent compounds.
  • FIG. 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.
  • FIG. 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.
  • Pstl the non-methylation-sensitive restriction enzyme
  • Smal methylation-sensitive restriction enzyme
  • FIG. 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).
  • FIG. 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 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.
  • 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; where
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • a maternal blood sample is drawn, and the plasma is separated utilizing standard techniques.
  • Fetal nucleic acids such as RNA
  • the fetal nucleic acids 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.
  • 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 GENECHIPTM 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.
  • gene expression is determined for one or more of the following genes: CD81, CDKNlC, DCN, DIO3, DLKl, GATM, GNAS 5 GRBlO, HYMAI, IGF2, IGF2R, MEG3, MEST, MKRNl 5 NDN, NNAT 5 MESTO 5 PEG3, PLAGLl, PON2, PPPlCC, SGCE 5 SNRPN 5 SNURF, PHLDA2, UBE3A, ZIM2, AGTRl, CRH , DSCRl, GLRX, HPGD, IGFl, INDO 5 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • tissue samples 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.
  • nucleic acid extracted is RNA.
  • RNA may be extracted using TRIZOLTM (Invitrogen, Carlsbad, California).
  • 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).
  • 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.
  • PCR polymerase chain reaction
  • 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.
  • nucleic acids may be detected by hybridization with a complementary sequence, such as an oligonucleotide probe.
  • 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.
  • 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 GENECHIPTM, 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.
  • GENECHIPsTM 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.
  • 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-
  • Computer software may be utilized to perform the statistical analysis. Examples of computer software which may be used in the present invention include GENESPRINGTM (Agilent Technologies, Palo Alto, California) and STATCRUNCHTM (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.
  • 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.
  • 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.
  • 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 I 5 MEG8, UBE3A, ATPlOA, GATM 5 TCEB3C, ZIM3, GNAS, TSIX.
  • Paternally expressed and maternally repressed genes include, but are not limited to: DIRAS3, NAP1L5, HYMAI, PLAGLl, SGCE, PEGlO, PONl, MEST 5 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
  • IUGR-related 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.
  • 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.
  • 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.
  • 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, ABCGl 5 ABHD5, ACSL4, ADFP, ADK, AMDl, BCAR3, BCL6, BZW2, C14orf58, C6orf4, CAP2, CAPN6, CEBPB, CLTB, CMAH, COBLLl, CRIPl, CSF2RB, CYB5R1, CYP19A1, DKFZP564O123, EBI3, EFHDl, FDXl, FLJlO
  • 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 IUGR 5 i.e., have expression ratios which are less than I 5 include but are not limited to: ADAMTSL3, ClR 5 CD44, DKFZP586H2123, OLFML3, PTPRD 5 PTPRK 5 SLIT2, SRPX 5 STABl, THSDl, THYl, Z39IG, AOC3, BMP5, COLl 4Al, COL15A1, COL21A1, COL5A2, COL6A1, COL6A3, DEFAl, ENPP2, FMOD, HG4, IGFl 5 LAMA2, LAMC3 , LIPG, LOXL2, MMP2, NID 1 , PCOLCE 5 WFDC 1 , WNT2, AGTRl 5 CBFA2T
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 e.g., 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).
  • 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.
  • the reagent for purifying RNA from a tissue or fluid sample is TRIZOLTM.
  • 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.
  • the reagents and equipment for measuring gene expression includes a GENECHIPTM.
  • the GENECHIPTM may be constructed to detect expression of one or more of the gene sequences identified in Table 2.
  • the GENECHIPTM 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.
  • 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
  • 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.
  • RNALATERTM 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.
  • RNA from placental tissues pulverized under liquid nitrogen was prepared using TRIZOLTM (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 0 C overnight; washing was at 64 0 C in 0.1% SDS/0.1 x SSC.
  • Genomic DNA was prepared by SDS/Proteinase K lysis followed by phenol/chloroform extraction and ethanol precipitation. The purified DNA, 4 ⁇ g, 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).
  • 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).
  • RMA Robust Multi- Array Analysis
  • 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
  • PHLDA2 a.k.a. IPL, TSSC3, BWRlC
  • MEST paternally expressed/maternally repressed gene
  • cytotrophoblast Saxena et al. 2003 and Mayer et al. 2000
  • cytotrophoblast Saxena et al. 2003 and Mayer et al. 2000
  • 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).
  • 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.
  • preeclampsia a common pregnancy complication that is distinct from IUGR
  • PHLDA2/MEST mRNA ratio a common pregnancy complication that is distinct from IUGR
  • 18 carried a clinical diagnosis of preeclampsia.
  • 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.
  • 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.
  • DMR differentially methylated region
  • 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).
  • 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).
  • IPL affinity-purified polyclonal anti-PHLDA2
  • 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.
  • 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.
  • 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
  • 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.
  • PHLDA2 expression was increased and MEST, MEG3, GATM, GNAS and PLAGLl expression was decreased in IUGR.
  • 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.
  • 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.
  • 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
  • INHBA indoleamine-pyrrole 2,3-dioxygenase
  • INDO/IDO indoleamine-pyrrole 2,3-dioxygenase
  • IGFl insulin-like growth factor-1
  • GLRX glucose-like growth factor-1
  • 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
  • several metabolite transporter genes SLC-family facilitated diffusion channels
  • DSCRl 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
  • 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.
  • this same type of transcriptional response might be physiologically advantageous when the hypoperfusion is only regional within an otherwise normal placenta.
  • 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.
  • 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.
  • 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.
  • CDKNlC gene encoding a cyclin-cdk inhibitor
  • PHLD A2 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).
  • 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.
  • 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).
  • 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.
  • 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 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.
  • Efstratiadis Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith-Wiedemann and Simpson-Golabi-Behmel syndromes,
  • Wiedemann-Beckwith syndrome further prenatal characterization of the condition
  • VEGF Down syndrome critical region protein 1
  • DSCRl Down syndrome critical region protein 1
  • 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.
  • D. Dao CP. Walsh, L. Yuan, D.
  • Placenta weight (mean ⁇ S.D.)** 419.5 (162.1) 287.8 (103.6)
  • 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
  • 200636_s a PTPRF NM_00284 1 63 00028 receptor type, F 200636_s_ membrane p possible differentiation protein tyrosine phosphatase,
  • CSF-1 trophoblastic 209716_at CSF1 M37435 0.59 00000 precursor 209716_at angioge ⁇ esis 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
  • Affy ID Symbol Genbank IUGR T-tast Description Affy ID Category 2 ? Function adipose lipid diff ⁇ rentiallon- accumulation/
  • 201196_S a AMD1 M21154 1.67 O 0003 decarboxylase 1 201196_s_at breast cancer anti- estrogen resistance
  • HIV-1 Tat interactive protein 2 HIV-1 Tat interactive protein 2
  • 216321 s a NR3C1 X03348 059 00003 receptor 216321_s_. endocrine signaling receptor gene
  • nucleobindin 2 203675_at calcium binding calcium binding ornithine decarboxylase
  • 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 ga ⁇ glioside-induced differentiation- associated protein 1

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Abstract

Selon l'invention la restriction de croissance intra-utérine (RCIU) est l'un des états pathologiques obstétriques les plus courants et les plus coûteux. Les études hospitalières et les études de registres montrent de façon cohérente que la restriction de croissance intra-utérine (RCIU) prédispose à la mortalité, à la morbidité post-natale et à des complications ultérieures, y compris les bronchopneumopathies chroniques et aiguës, l'entérocolite nécrosante, les hémorragies intra-ventriculaires et la rétinopathie des prématurés. La présente invention concerne une méthode d'identification des gènes qui contribuent à la restriction de croissance intra-utérine (RCIU), une méthode de diagnostic de RCIU chez un sujet et des kits de diagnostic de RCIU chez un sujet.
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WO2009034353A1 (fr) * 2007-09-14 2009-03-19 Leeds Teaching Hospitals Nhs Trust Puce à empreinte
CN106512009A (zh) * 2016-10-28 2017-03-22 武汉大学 Ph同源域家族a成员3（phlda3）在治疗心肌肥厚中的应用
CN109913537A (zh) * 2019-03-21 2019-06-21 首都医科大学附属北京朝阳医院 一种牛磺酸对iugr胎鼠神经干细胞增殖的应用方法
WO2019222812A1 (fr) * 2018-05-24 2019-11-28 The University Of Melbourne Biomarqueurs circulatoires pour la santé placentaire ou fœtale
WO2020011195A1 (fr) * 2018-07-13 2020-01-16 立森印迹诊断技术有限公司 Modèle de classement utilisé pour détecter un degré de malignité et de bénignité de tumeurs pulmonaires et application correspondante
WO2020098604A1 (fr) * 2018-11-12 2020-05-22 立森印迹诊断技术有限公司 Modèle de classification permettant de déterminer le caractère bénin ou malin des tumeurs prostatiques et utilisation correspondante
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EP3452593B1 (fr) * 2016-05-02 2020-12-23 Medizinische Hochschule Hannover Lncrna meg3 pour la thérapie et le diagnostic du remodelage cardiaque
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CN117079823A (zh) * 2023-10-17 2023-11-17 北京大学第三医院（北京大学第三临床医学院） 早期预测选择性胎儿生长受限发病风险筛查的系统和方法

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2465948A (en) * 2007-09-14 2010-06-09 Leeds Teaching Hospitals Nhs Trust Imprinted array
WO2009034353A1 (fr) * 2007-09-14 2009-03-19 Leeds Teaching Hospitals Nhs Trust Puce à empreinte
EP3452593B1 (fr) * 2016-05-02 2020-12-23 Medizinische Hochschule Hannover Lncrna meg3 pour la thérapie et le diagnostic du remodelage cardiaque
US11186838B2 (en) 2016-05-02 2021-11-30 Medizinische Hochschule Hannover LNCRNA MEG3 for therapy and diagnosis of cardiac remodelling
CN106512009A (zh) * 2016-10-28 2017-03-22 武汉大学 Ph同源域家族a成员3（phlda3）在治疗心肌肥厚中的应用
CN106512009B (zh) * 2016-10-28 2019-10-11 武汉大学 Ph同源域家族a成员3（phlda3）在治疗心肌肥厚中的应用
WO2019222812A1 (fr) * 2018-05-24 2019-11-28 The University Of Melbourne Biomarqueurs circulatoires pour la santé placentaire ou fœtale
WO2020011195A1 (fr) * 2018-07-13 2020-01-16 立森印迹诊断技术有限公司 Modèle de classement utilisé pour détecter un degré de malignité et de bénignité de tumeurs pulmonaires et application correspondante
WO2020098604A1 (fr) * 2018-11-12 2020-05-22 立森印迹诊断技术有限公司 Modèle de classification permettant de déterminer le caractère bénin ou malin des tumeurs prostatiques et utilisation correspondante
WO2020103905A1 (fr) * 2018-11-21 2020-05-28 立森印迹诊断技术有限公司 Marqueurs pronostiques dans le cancer du poumon, modèle de typage pronostique du cancer du poumon et application correspondante
CN109913537A (zh) * 2019-03-21 2019-06-21 首都医科大学附属北京朝阳医院 一种牛磺酸对iugr胎鼠神经干细胞增殖的应用方法
CN109913537B (zh) * 2019-03-21 2022-07-08 首都医科大学附属北京朝阳医院 一种牛磺酸对iugr胎鼠神经干细胞增殖的应用方法
WO2021018116A1 (fr) * 2019-07-30 2021-02-04 立森印迹诊断技术有限公司 Marqueur tumoral et son utilisation
EP4006171A4 (fr) * 2019-07-30 2023-08-09 Lisen Imprinting Diagnostics Wuxi Co., Ltd Marqueur tumoral et son utilisation
CN116287187A (zh) * 2022-12-21 2023-06-23 上海市生物医药技术研究院 miR-3074-5p作为宫内生长受限标志物和治疗靶点的应用
CN117079823A (zh) * 2023-10-17 2023-11-17 北京大学第三医院（北京大学第三临床医学院） 早期预测选择性胎儿生长受限发病风险筛查的系统和方法
CN117079823B (zh) * 2023-10-17 2024-01-02 北京大学第三医院（北京大学第三临床医学院） 早期预测选择性胎儿生长受限发病风险筛查的系统和方法

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