WO1999043851A1 - Gene de susceptibilite a la pre-eclampsie et aux fausses-couches lie au hla - Google Patents

Gene de susceptibilite a la pre-eclampsie et aux fausses-couches lie au hla Download PDF

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WO1999043851A1
WO1999043851A1 PCT/IE1999/000012 IE9900012W WO9943851A1 WO 1999043851 A1 WO1999043851 A1 WO 1999043851A1 IE 9900012 W IE9900012 W IE 9900012W WO 9943851 A1 WO9943851 A1 WO 9943851A1
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hla
eclampsia
cells
dna
miscarriage
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PCT/IE1999/000012
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English (en)
Inventor
Margaret O'brien
John Bermingham
Kathleen A. Quane
David M. Jenkins
Tommie V. Mccarthy
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National University Of Ireland, Cork
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Priority to JP2000533590A priority Critical patent/JP2003517267A/ja
Priority to AU26363/99A priority patent/AU2636399A/en
Priority to CA002321223A priority patent/CA2321223A1/fr
Priority to EP99906418A priority patent/EP1056886A1/fr
Publication of WO1999043851A1 publication Critical patent/WO1999043851A1/fr
Priority to NO20004222A priority patent/NO20004222L/no
Priority to AU2003231618A priority patent/AU2003231618A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/06—Antiabortive agents; Labour repressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12Q2600/172—Haplotypes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a susceptibility gene for pre-eclampsia and eclampsia, and the use of such a gene in methods for diagnosing susceptibility to these diseases
  • the invention also relates to a test kit for diagnosis of susceptibility and to pharmaceutical compositions for the prevention or treatment of the diseases
  • the invention can also be used in the diagnosis of susceptibility to miscarriage and/or miscarriage-related infertility and/or lntraute ⁇ ne growth retardation
  • the present invention relates to methods and materials used to detect a HLA linked human pre-eclampsia and miscarriage predisposing gene (HLA-G), some alleles of which, or iinked alleles of linked genes of which, cause susceptibility to pre-eclampsia and miscamage More specifically, the invention relates to sequence va ⁇ ation in the HLA-G gene and linked genes and their use in the diagnosis of susceptibilit y to pre-eclampsia and miscarriage The invention further relates to sequence variations in the HLA-G gene and their use in diagnosis and prognosis of pre-eclampsia and miscarriage Additionally the invention relates to the therapy for pre-eclampsia and miscarriage and for susceptibility to pre- eclampsia and miscarriage including protein therapy, gene therapy and protein mimetics The invention also relates to screening for drugs for pre-eclampsia and miscarriage therapy and for susceptibility to to pre
  • Pre-eclampsia is the major cause of foetal and maternal morbidity and mortality with probable long term adverse effects on health due to the prolonged associated mtraute ⁇ ne hypoxia
  • Pre-eclampsia occurs m approximateh five to ten percent of all population births and is uniquely a disease of pregnancv Acute pathological changes begin to resolve soon after delivery
  • the pathologic mechanisms causing pre- eclampsia are unclear and no marker predictive for the disease prior to clinical evidence of the disease has been identified Furthermore an association has been observed between miscamage and pre-eclampsia (Cooper et al . 1988)
  • pre-eclampsia Although the cause of pre-eclampsia is unknown, hypertension is observed in pre-eclampsia and has been the focus of a large amount of research on the disorder However, the pathological and physiological changes of pre-eclampsia show that this syndrome is much more than pregnancy-induced hypertension Evidence to date implicates the action of placental trophoblasts as the underlying cause
  • cytotrophoblast invasion is shallow and spiral artenol invasion is abnormal, resulting m reduced blood perfusion of the intervillous space Moreover the characteristic pattern of integ ⁇ n switching that takes place during normal trophoblast differentiation does not occur in pre-eclampsia
  • the outermost layer (trophoblasts) of the human placenta is devoid of classical class I human leukocyte antigens (HLA-A and HLA-B) and class II proteins (HLA-DR, HLA-DQ and HLA-DP) Although this prevents recognition by maternal T lymphocytes, the lack of class I molecules leaves these cells susceptible to attack by natural killer (NK) cells.
  • trophoblast cells directly in contact with maternal tissues selectively express a characte ⁇ stic nonclassical class lb molecule.
  • HLA-G HLA-E and limited HLA-C expression also occurs Expression of HLA-G has been shown to be sufficient to protect otherwise susceptible target cells from NK cell mediated lysis NK cells usually express several different inhibitory receptors of various specificities at the same time Cross linking of any single inhibitory receptor is sufficient to inactivate NK cell activity against all possible targets It has been shown that membrane bound HLA-G molecules were able to inhibit alloreactive NK cells with NK inhibitory receptor 1 and inhibitory receptor 2 (NK1 and NK2) It has been shown that CD94 / NKG2 is the predominant inhibitory receptor involved in recognition of HLA-G by decidual and peripheral NK cells Thus, at a functional level, HLA-G is able to protect target cells from destruction by NKl-,NK2-and NKG2 specific effector cells (Loke and King.
  • HLA-G has been shown to modulate the ability of blood mononuclear cells to release cvtokines (Maejima et al 1997) suggesting a role for HLA-G in triggering maternal -foetal immune interplay Specifically, cocultu ⁇ ng of HLA-G expressing cells with 3 peripheral blood mononuclear cells (PBMC) increased the amount of ⁇ nterleukm-3 (IL-3) and mterleukin- 1 beta (IL-1 beta) and decreased the amount of tumour necrosis factor-alpha (TNF-alpha) release from the PBMC cells
  • PBMC peripheral blood mononuclear cells
  • HLA-G binds a diverse but limited array of peptides in a manner similar to that found for classical class I molecules and it has been reported that HLA-G is expressed m the human thymus raising the possibility that maternal unresponsiveness to HLA-G expressing foetal tissues may be shaped in the thymus by central presentation of this MHC molecule on the medullary epithelium (C ⁇ sa et al 1997) HLA-G is known to be capable of stimulating a HLA-G restricted cytotoxic T lymphocyte response and HLA-G molecules can serve as target molecules in lytic reaction with cytotoxic T lymphocytes and HLA-G expressed internally in vivo in transgenic animals is involved in education of the lymphocytic repertoire (Schmidt et al , 1997)
  • Maior histocompatibility (MHC) molecules bind a diverse array of peptides for presentation to T cells as part of a mechanism for recognition of self and non-self cells and pathologically altered cells
  • MHC Maior histocompatibility
  • HLA bound peptides can readily be fractionated, fully or partially purified and sequenced and can be assayed for their capacity to promote an immune reaction by measurement of their capacity to reconstitute lysis of target cells by cytotoxic T cells (den Haan et al , 1998)
  • HLA-G The entire gene sequence of HLA-G is known and DNA sequence analysis of HLA-G has shown that the HLA-G gene exhibits limited polymorphism van der Van & Ober. 1995 examined the first six exons of HLA-G in 45 healthy African-Americans and observed variations in exons 2 and 3, which correspond to the alpha 1 and alpha 2 domains of the peptide binding grove The most common polymorphism observed was a C to T transition at position 1488, corresponding to codon 93 Another common pol morphism was identified by Harrison et al.
  • HLA-G is a polymorphic gene potentially capable of presenting a wide variety of peptides Patterns of variability in HLA-G are similar to those of other class I MHC genes, where amino acid substitutions are clustered in the alpha 1 and alpha 2 domains
  • Diagnosis of true pre-eclampsia can be complicated by other hypertensive disorders such as essential hypertension and hypertension arising from renal disease Such hypertensive disorders are distinct from true pre-eclampsia but nonetheless can confound diagnosis and thus pose problems for genetic studies
  • pre-eclampsia The classification of pre-eclampsia by some investigators as a disease of immune dysfunction has prompted a number of studies on the role of the major histocompatibility complex in the genetics of pre- eclampsia
  • HLA-G deletion polymorphism for association with pre- eclampsia Specifically, pre-eclamptic patients, offspring of pre-eclamptic mothers, blood relatives of pre- eclamptic patients, husbands of pre-eclamptic patients and a normal control group were genotyped for the polymorphism The was no detectable association between pre-eclampsia in mothers or in offspring of pre-eclamptic mothers and the HLA-G deletion polymorphisms
  • pre-eclampsia In the largest study of monozygotic twins, pre-eclampsia was reported in five first pregnancies, and all affected mothers were discordant with their twin A second well documented report on an identical set of twins also showed clear discordance for pre-eclampsia in their first pregnancies
  • HLA-G genotypes in pre- eclamptic and control trios we have investigated HLA-G genotypes in pre- eclamptic and control trios and have shown that HLA-G is linked to both normal and pre-eclampsia pregnancv outcome and associated with recurrent spontaneous abortion
  • HLA- G genotypes in second pregnancies of control and pre-eclamptic trios have shown that the presence of specific HLA-G alleles in the foetus in first pregnancy permits the occurrence of different HLA-G alleles in second pregnancy showing that HLA-G can induce tolerance to antigens in the first pregnancy and/or can modify the maternal immune system to accept foetuses in the second pregnancy in the
  • Recurrent spontaneous abortion or recurrent miscarriage, defined as the loss of three or more spontaneous pregnancies before 20 weeks gestation, occurs in less than 1% of pregnant women
  • RSA Recurrent spontaneous abortion
  • RSA is a condition with many different causes, however, about 50% of all RSA cases are not explained by structural genetic, endocrine, infectious or anatomic factors
  • recurrent pregnancy loss may have autoimmune (immunity against self) and alloimmune (immunity against another person) causes, even in women with no clinically diagnosed autoimmune diseases This has lead to investigation of the role of the HLA system and RSA.
  • HLA-DR sharing in couples with RSA and also a significant excess of HLA-DQ sharing in couples with unexplained infertility
  • a method for diagnosing susceptibility to normal pregnancv pre-eclampsia and or eclampsia and/or lntrauterme growth retardation and/or susceptibility to miscarriage and/or miscarriage-related infertility comprising the steps of a) obtaining a fluid and/or tissue sample from a female and or male and/or foetus and either b) determining the sequence of all or part of the HLA-G nucleic acid, and/or HLA-G linked nucleic acid or c) detecting variant forms of all or part of the HLA-G protein, and/or proteins encoded b ⁇ HLA-
  • the HLA-G nucleic acid is analysed by the presence of the C and/or T allele of codon 93 in exon 3 and/or the insertion and/or deletion allele of exon 8
  • the effect of one or more of the HLA-G sequence variants on the functional activity of HLA-G and or on the size and or the level of all or part of the HLA-G mRMA and/or its encoded peptide is measured
  • the present invention provides a method of diagnosing susceptibility to pre-eclampsia and or eclampsia and/or intrauterine growth retardation a nd/or susceptibility to miscarriage and/or miscarriage-related infertility comprising the steps of a) obtaining nucleic acid from a parent and/or a prospective parent and/or foetus, b) establishing the HLA-G sequence variants present in the parent and or foetus by analysing the nucleic acid isolated in step (a), and c) comparing the HLA-G sequence variants identified in step (b) with known HLA-G sequence variants
  • the HLA-G sequence variants are established by characterising all or part of the DNA sequence of the HLA-G gene by methods selected from DNA sequencing. PCR-restnction fragment length polymorphism analysis, glycosylase mediated polymorphism detection, oligonucleotide hybridisation, gel electrophoretic detection of polymo ⁇ hisms and amplification based detection approaches
  • the invention also provides a test kit for the diagnosis of susceptibility to normal pregnancy, pre- eclampsia and/or eclampsia and/or intrauterine growth retardation and/or susceptibility to miscarriage and/or miscarriage-related infertility comprising a) oligonucleotide primers for amplification of all or part of the HLA-G gene and/or HLA-G linked DNA. b) amplification reagents for amplification of genomic DNA and/or RNA segments, selected from a DNA / RNA polymerase. a reverse transc ⁇ ptase. the deoxy ⁇ bonucleotides dATP. dCTP. dGTP.
  • dTTP and dUTP and or ⁇ bonucleotides ATP, CTP, GTP, TTP and UTP, and reaction buffer, c) reagents for identifying sequence variants in DNA and/or RNA d) control DNA and or RNA
  • the primers of (a) allow specific amplification of all or part of the HLA-G gene using the polymerase chain reaction
  • C/T-93 A C to T polymorphism occurs at nucletide 1488 in the third position of codon 93 and is referred to as C/T-93 herein where C-93 is one allele of the polymorphism and T-93 is the other allele of the polymorphism
  • the C/T-93 polymorphism is genotyped by PCR amplification of a section of intron 2 - exon 3 using the primers 5 " -TACTCCCGAGTCTCCGGGTCTG-3 (SEQ ID NO 1) as the forward pnmer and 5 -AGGCGCCCCACTGCCCCTGGTAC-3 '(SEQ ID NO 2) as the reverse primer giving rise to the amplified C-93 allele (SEQ ID NO 4) and amplified T-93 allele (SEQ ID NO 5) followed by by by
  • the common 14 base pair insertion / deletion polymorphism in exon 8 of the HLA-G gene is referred to as I/D-E8 herein where I-E8 is one allele of the polymorphism and D-E8 is the other allele of the polymorphism
  • genotypmg of the HLA-G exon 8 deletion polymorphism is performed by amplifying a short section flanking the deletion location in exon 8 This is achieved using the polymerase chain reaction with primers designed to hyb ⁇ dise to known DNA sequence in exon 8
  • the forward primer is 5 * -TGTGAAACAGCTGCCCTGTGT-3' (SEQ ID NO 10) and the reverse pnmer is 5'- AAGGAATGCAGTTCAGCATGA-3 * (SEQ ID NO 11)
  • the I D exon 8 polymorphism is genotyped bv size separation of the PCR products on a 10% non denaturing polyacrylamide gel and visualised by staining with ethidium bromide, the I
  • allele specific genotypmg is performed in cases where maternal and paternal C/T-93 and I/D- E8 HLA-G haplotypes cannot be directly assigned This is achieved using allele specific primers which allows selective amplification of the I-E8 or D-E8 allele Following allele specific amplification, the C/T- 93 polymorphism is then genotyped using the GMPD assay described above
  • Primers for amplification of the I-E8 allele are 5 -TACTCCCGAGTCTCCGGGTCTG-3' (SEQ ID NO 1) as the forward primer and 5 -CAAAGGGAAGGCATGAACAAATCTTG-3' (SEQ ID NO 14) as the reverse primer
  • Primers for amplification of the D-E8 allele are 5--TACTCCCGAGTCTCCGGGTCTG-3 (SEQ ID NO 1) as the forward primer and 5 -GTTCTTGAAGTCACAAAGGGACTTG -3 " (SEQ ID NO 15) as the reverse primer
  • SEQ ID NO 15 the reverse
  • haplotypes are constructed and suitably, transmitted and non-transmitted alleles to offspring / foetus are assigned
  • the amplification reagents include a thermostable DNA polymerase, amplification buffer and DNA precursor nucleotides
  • HLA-G sequence and/or HLA-G linked sequence may also be amplified, by a method or combination of methods selected from nucleic acid sequence based amplification, self-sustained sequence replication, transcription-mediated amplification, strand displacement amplification and the ligase chain reaction
  • the comparison of one or more variants identified is performed by association and/or linkage analaysis and/or transmission analysis
  • all or part of the HLA-G sequence is cloned into a vector
  • the invention may involve a) obtaining nucleic acid or fluid or tissue sample from a parent and/or prospective parent and/or foetus. b) establishing the HLA genotype or serotype of the parent/prospective parent and/or foetus by analysing the nucleic acid or fluid or tissue sample isolated in step (a), c) comparing the HLA genotypes or serotypes identified in step (b) with known HLA genotypes or serotypes respectively
  • the method involves the measuring of cellular and/or soluble HLA-G levels
  • HLA- G levels are measured by immunoassay using an antibody for specific HLA-G protein
  • the invention may involve identifying the variant form of HLA-G protein and/or the levels thereof present in the sample
  • HLA-G variant proteins and/or levels thereof are detected and/or quantified by immunoassay using specific antibodies which detect HLA-G variants, or HLA-G protein
  • antibody specific for HLA-G protein variants and/or electrophoretic separation methods and/or chromatographic separation methods may be used
  • Preferred methods for detecting HLA-G protein and variants thereof include, enzyme linked immunosorbent assays (ELISA), radioimmuno-assav s (RIA), immunoradiometnc assays (IRMA) and immunoenzymatic assays (IEMA). including sandwich assays using monoclonal and/or polyclonal antibodies 1 1
  • the invention may involve measuring of level of molecules whose concentration changes as a direct and/or indirect result of HLA-G action
  • the molecules are selected from IL-1. IL-2. IL-3, IL-4. IL-6. IL- 10 beta and tumour necrosis factor alpha
  • the levels of such molecules are measured bv immunoassay using antibodies specific for the molecules
  • the method may involve measuring of levels of trophoblast specific markers
  • the trophoblast markers are cytokeratins pregnancy specific glycoprotem 1.
  • human cho ⁇ onic gonadotrophin and human placental lactogen Preferably the levels of such molecules are measured by immunoassay using antibodies specific for the molecules
  • the method may comprise the steps of a) incubating blood mononuclear cells and/or a subset of such cells with one or more HLA-G variants and/or any combination thereof and/or cells expressing all or part of one or more variants of the HLA-G gene and/or a combination of one or more variants thereof, wherein the blood mononuclear cells and/or
  • HLA-G variant is from a female and/or male and/or foetus b) analysing the activity of the blood mononuclear cells and/or the HLA-G and/or cells expressing one or more HLA-G variant
  • the blood mononuclear cells are obtained as a blood sample and/or tissue sample from the female and/or are obtained through matching the females blood mononuclear cells with blood mononuclear cells from a donor and/or cell line panel
  • populations of T cells and or NK cells are isolated from the blood sample by density cent ⁇ fugation and/or immunoselection
  • blood mononuclear cells matching the females blood mononuclear cells are identified from a test panel by matching the HLA serotype and or extended HLA genotype and/or HLA-G genotype of the female with the HLA serotype and/or extended HLA genotype and/or HLA-G genotype of blood mononuclear cells Preferably.
  • HLA-G matching the male and/or female HLA-G is identified from a test panel by matching the HLA-G type and/or HLA-G genotype of the male and/or female with the HLA-G type and/or HLA-G genotype of HLA-G proteins and/or cells expressing one or more HLA-G gene variants in the test panel
  • a test panel is assembled by growing cells expressing one or more HLA-G variants
  • Such cells mav be derived from natural tissue such as placenta and or created artificially by the introduction of one or more vectors bearing HLA-G gene variants which are capable of promoting the expression of the HLA-G gene into a cell and or by inducing the expression of native HLA-G in cells
  • the vector used is plasmid.
  • HLA-G protein is used as a crude preparation and/or fully or partially purified from such cells
  • HLA-G protein may be loaded with binding peptides naturally or artificially
  • the HLA-G - blood mononuclear cell interaction is measured by assessing blood mononuclear cell activation including assessment of one or more of the following, cell proliferation, transformation cytotoxic response, surface marker expression, cytokine production, conjugate formation and target specificity
  • the method may comprise the steps of a) cloning the HLA-G gene from a parent and/or prospective parent and/or foetus, b) expressing the HLA-G protein from the cloned gene in vitro and/or in vivo, c) measuring the levels of activity of the expressed HLA-G protein, d) comparing the levels of activity of the expressed HLA-G protein with the levels of activity observed for the normal HLA-G protein
  • the method may also comprise a) establishing all or part of the HLA-G sequence and/or HLA-G linked sequences present in a sample from a female and/or male and/or foetus by analysing the nucleic acid from said sample, b) determining whether one or more of any variants or any combination thereof, identified in step (a) are indicative of susceptibility to normal pregnancy or pre-eclampsia and/or eclampsia and/or intrauterine growth retardation and/or susceptibility to miscarriage and/or miscarriage-related infertility by comparative analysis and/or analysis of the effect of one or more of the variants on the functional activity of HLA-G and/or on HLA-G mRNA
  • the HLA-G sequence variants are established by characterising all or part of the DNA sequence of the HLA-G gene and/or closely linked DNA including HLA-A, HLA-B, HLA-C. HLA-E, HLA-F and HLA-H genes by amplifying all or parts HLA-G or closely linked DNAs and identifying the sequence variants present using one or more sequence variation detection methods
  • one or more copies of all or parts of the HLA-G gene is amplified by any of several amplification approaches such as the polymerase chain reaction (PCR). nucleic acid sequence based amplification (NASBA). self sustained sequence replication (3SR). transcription-mediated amplification (TMA) and strand displacement amplification Amplification of a target nucleic acid molecule may also be carried out using a the ligase chain reaction (LCR) and a variation of the LCR which employs a short PCR step (PLCR) Suitably, DNA or mRNA is used as the amplification substrate Suitably.
  • PCR polymerase chain reaction
  • NASBA nucleic acid sequence based amplification
  • SR self sustained sequence replication
  • TMA transcription-mediated amplification
  • PLCR short PCR step
  • DNA sequence variations are detected by any one or more of a variety of gene variation detection methods including DNA sequencing, glycosylase mediated polymorphism detection, restriction fragment length polymorphism analysis, enzymatic or chemical cleavage assays, hybridisation to DNA probe arrays, allele specific oligonucleotide hybridisation assays, allele specific amplification methods such as the amplification refractory method (ARMS), electrophoretic detection of polymorphisms based on migration through a gel matnx. 5 " nuclease assay and ligase chain reaction
  • one or more variants identified are known vanants associated with susceptibility to normal pregnancy and or susceptibility to pre-eclampsia and/or eclampsia and/or intrauterine growth retardation and/or susceptibility to miscarriage and/or miscarriage-related infertility
  • comparative analysis is performed by gene association and/or gene linkage methods to determine whether HLA-G variants and/or HLA-G linked variants are associated with normal pregnancy and/ or susceptibility to pre-eclampsia and/or eclampsia and/or intrauterine growth retardation and/or susceptibility to miscarriage and/or miscarriage-related infertility
  • HLA-G va ⁇ ants associated with normal pregnancy and/ or susceptibility to pre-eclampsia and/or eclampsia and/or intrauterine growth retardation and/or susceptibility to miscarriage and/or miscarriage-related infertility can be identified by the effect of HLA-G variants on HLA-G function Suitably.
  • HLA-G variants are functionally analysed by measuring the interaction of one or more of the HLA-G variants and/or any combination thereof, with blood mononuclear cells and/or measuring the size and level of the HLA-G messenger RNA and/or the size and level of HLA-G gene product and/or peptide binding for one or more of the HLA-G variants and/or any combinations thereof
  • HLA-G - blood mononuclear cell activity is measured by assessing blood mononuclear cell activation including assessment of one or more of the following, cell proliferation, cytotoxic response, surface marker expression, cytokme production, conjugate formation and target specificity
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically effective amount of HLA-G protein and/or cells expressing HLA-G and/or one or more peptides which binds to HLA-G and/or blood mononuclear cells from a donor and/or a cells from a test panel known to interact with HLA-G variants, cytokines and any combination thereof including IL-1 beta , IL-2. IL-3. IL-4, IL-6. IL-10 and tumour necrosis factor-alpha and/or inhibitors of cytokines and/or tumour necrosis factor alpha and/or derivatives of cytokines and/or tumour necrosis factor-alpha, optionally with pharmaceutically-acceptable carriers or excipients
  • the invention also provides a method for screening for agents which can potentially be used as diagnostic indicators and/or drug targets for pre-eclampsia, miscarriage, miscarriage-related infertility and intrauterine growth retardation by a) measuring the expression level of one or more genes and / or proteins in HLA-G expressing cells and /or blood mononuclear cells and / or T cell and /or natural killer cell subsets thereof following interaction with HLA-G and / or HLA-G expressing cells, 14 b) comparing the expression level identified in step (a) with the expression level in HLA-G expressing cells and /or the blood mononuclear cells and / or T cell and /or natural killer cell subsets thereof following interaction with HLA-G and / or HLA-G expressing cells in normal pregnancy and/ or pre- eclampsia pregnancy and/or intrauterine growth retardation pregnancv and/or miscamage pregnancv and/or miscarriage-related in
  • the invention provides a method for screening for potential pre-eclampsia and eclampsia and intrauterine growth retardation and miscarriage and miscarriage-related infertility therapeutic agents selected from a) identifying agents which alter the expression of HLA-G.
  • identifying agents which alter the activity of HLA-G c) identifying agents which mimic the action of HLA-G, d) identifying agents which bind to HLA-G, e) identifying peptides which bind to HLA-G, f) identifying agents which bind to HLA-G receptors
  • identifying expressed genes using DNA probe arrays in a cellular background whose expression is altered in response to HLA-G expression in the cells and/or m response to interacting cells expressing HLA-G.
  • sperm and/or semen and/or female reproductive tissue are screened for agents a) which alter the expression of HLA-G in fertilised eggs and/or embryos, b) which alter the cell cleavage rate of fertilised eggs and/or embryos c) which induce cellular factors in cell in culture and/or cell in vivo that alter the cell cleavage rate of fertilised eggs and/or embryos
  • the method may involve a) measuring the expression level of one or more genes and/or proteins in HLA-G expressing cells and/or blood mononuclear cells and/or T cell and/or natural killer cell subsets thereof following interaction with HLA-G and/or HLA-G expressing cells, b) comparing the expression level identified in step (a) with the expression level in HLA-G expressing cells and/or the blood mononuclear cells and/or T cell and/or natural killer cell subsets thereof following interaction with HLA-G and/or HLA-G expressing cells associated with normal pregnancv and/ or pre-
  • SUBST ⁇ UTE SHEET (RULE 26) 15 eclampsia pregnancy and/or intrauterine growth retardation pregnancy and/or miscarriage pregnancy and/or miscarriage-related infertility
  • blood mononuclear cells and/or HLA-G expressing cells are obtained from a female and/or male and/or foetus and/or test panel of blood mononuclear cells and/or HLA-G expressing cells
  • gene expression is measured by any one or combination of several methods including hybridisation between cDNA and/or RNA from the cells and DNA probes and/or RNA probes and/or DNA probe arrays, quantitative amplification approaches such as quantitative (reverse transc ⁇ ptase - polymerase chain reaction) RT-PCR.
  • quantitative amplification approaches such as quantitative (reverse transc ⁇ ptase - polymerase chain reaction) RT-PCR.
  • protein expression is measured by any one or combination of several methods including one dimensional and or two dimensional gel electrophoresis and staining of proteins and/or detection of one or more proteins using, enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometnc assays (IRMA) and immunoenzymatic assays (IEMA). including sandwich assays and Western blotting using monoclonal and/or polyclonal antibodies
  • the method may involve a) measuring the expression level of one or more genes and/or proteins in cells expressing HLA-G, and b) comparing the expression level identified m step (a) with the expression level in HLA-G non- expressing cells
  • the cells are fertilised animal eggs and/or animal embryos
  • the invention also provides a method for the prevention of pre-eclampsia and/or eclampsia and/or intrauterine growth retardation and/or susceptibility to miscarriage and/or miscarriage-related infertility selected from a) treatment of a female with all or part of a pharmaceutically effective amount of an effective HLA-G protein and/or peptides which bind to HLA-G and/or cells expressing HLA-G, b) treatment of a female with all or part of a pharmaceutically effective amount of molecules or inhibitors of molecules whose level or activity is directly or indirectly altered by HLA-G.
  • the invention may compnse a) obtaining blood mononuclear cells and/or T cell and/or natural killer cell subsets thereof and/or HLA-G and/or HLA-G expressing cells from a female and/or male and/or foetus and/or test panel, b) measuring the expression level of one or more genes and/or proteins in the HLA-G expressing cells and/or blood mononuclear cells following interaction with HLA-G and/or HLA-G expressing cells, c) companng the expression level identified in step (b) with the expression level in the blood mononuclear cells and/or HLA-G expressing cells in normal pregnancy and/ or pre-eclampsia pregnancy and/or intrauterine growth retardation pregnancy and or miscarriage pregnancy and/or miscarriage-related infertility
  • the blood mononuclear cells and/or HLA-G expressing cells are obtained as a blood sample and/or tissue sample
  • populations of T cells and/or NK cells are isolated from the blood sample by density cent ⁇ fugation and/or immunoselection
  • HLA-G expressing cells are isolated by immunoselection
  • the invention also provides a method for improving fertility and pregnancy outcome wherein male and/or female partners and/or sperm and/or ova and/or recipients of fertilised eggs and/or zygotes / and/or embryos are selected on the basis of HLA-G so that their genotypes and/or serotypes are associated with normal pregnancy outcomes and/or not associated with pre-eclampsia and/or eclampsia and/or intrauterine growth retardation and/or susceptibility to miscarriage and/or miscarriage-related infertility
  • a method for improving pregnancy success selected from a) pre-treating the female with sperm and/or attenuated forms thereof, and/or semen and/or fractions thereof from a male with a known HLA-G genotype, prior to mating with a male of a different HLA-G genotype, and/or in vitro fertilisation using sperm from a male of a different HLA-G genotype and/or embryo transfer where the male HLA-G is of a different HLA-G genotype, b) mixing sperm of a known HLA-G genotype with sperm and/or attenuated forms thereof, and/or semen and/or fractions thereof from a male with a different HLA-G genotype prior to in vitro fertilisation
  • fertility and/or pregnancy outcome are improved by selection of male and / or female partners and / or sperm and / or ova and / or recipients of fertilised eggs and / or zygotes / and / or embryos so
  • Cloning of all or part of one or more HLA-G genes in any of the above methods may be achieved by amplification of all or part of one or more HLA-G genes and insertion of all or part of the amplified product into a vector capable of expressing the inserted gene
  • Expression of the HLA-G protein from the cloned gene in any of the above methods may be achieved by introduction of the expression vector into a suitable host such as a bacterium or an eukaryotic cell in culture
  • the level of activity of the expressed HLA-G protein in any of the above methods may be achieved by a) directly and/or indirectly measuring the interaction of the HLA-G protein and/or cells expressing HLA-G protein with blood mononuclear cells and/ or b) detecting one or more molecules whose level is altered as a result of the interaction of the HLA- G protein and/or cells expressing HLA-G protein with blood mononuclear cells and/or c) measuring changes in cell cleavage rate due to direct and/or
  • HLA-G as defined herein refers to any form of HLA-G and / any complex involving HLA-G including different isoforms of HLA-G arising from alternative splicing pathways, combination of different HLA-G isoforms secreted HLA-G.
  • membrane bound HLA-G HLA-G with peptides bound and HLA-G associated with beta -2-microglobuhn HLA-G protein refers to any cmde, partially and/or fully purified form of HLA-G
  • the invention also provides use of a DNA sequence selected from any one of sequence I D s 1 to 21 for diagnosis of susceptibility to or in a test kit for the diagnosis of susceptibility to normal pregnancy, pre- eclampsia and/or eclampsia and/or intrauterine growth retardation and/or susceptibility to miscarriage and/or miscarriage-related infertility, for monitoring progress of pregnancy, for use in the manufacture of a medicament in a method for screening potential therapeutic agents, in a method for screening for potential diagnostic indicators and/or drug targets, in a method for improving pregnancy success or in a method for the prevention of pre-eclampsia and/or eclampsia and/or intrauterine growth retardation and/or susceptibility to miscarriage and/or miscarriage-related infertility, for monitoring progress of pregnancv
  • the invention also provides a method for induction of tolerance in a host to a non-self tissue which comprises administering HLA-G and /or HLA-G loaded with peptides from the non-self tissue and /or HLA-G expressing cells derived from or related to the non-self tissue, and/or a non-self tissue bearing an introduced HLA-G gene so that HLA-G is expressed in all or part of the tissue
  • the invention provides a method for the treatment of autoimmune disease which comprises administering HLA-G and /or HLA-G loaded with peptides from a self or non-self tissue and / or with specific autoimmune antigen and /or HLA-G expressing cells from a self and/or non-self tissue and/or a self and or non-self tissue bearing an introduced HLA-G gene so that HLA-G is expressed in all or part of the tissue
  • pre-eclamptic patients were identified as p ⁇ magravidas who were delivered by caesarean section at or prior to 36 weeks gestation because of a deterioration in pregnancy indicative of pre-eclampsia Diagnostic symptoms were a rise m blood pressure >15mm Hg diastohc or >30mm Hg systolic from measurement in early pregnancy or to >140/90mm Hg in late pregnancy, and one or more of the following proteinu ⁇ a, odema, headache, visual disturbance, epigastric pain Control patients were identified as primagravidas with normal delivery and normal blood pressure 5-10mls of blood were taken from the offspring of p ⁇ magravida pre-eclampsia and normal pregnancies with informed consent
  • Genomic DNA was extracted from peripheral blood samples and/or cheek swab samples by standard methods DNA concentration was determined by absorbance at 260nm for samples where DNA was isolated from blood The integrity and punty of the genomic DNA was determined by agarose gel electrophoresis and OD260 OD280 ratio respectively
  • the C-93T HLA-G polymo ⁇ hism is also known as the C/T codon 93 polymo ⁇ hism (and as HLA-G C1488T) and referred to as C/T-93 herein where C-93 is one allele of the polymo ⁇ hism and T-93 is the other allele of the polymo ⁇ hism
  • exon 3 of the HLA-G gene was first amplified using the polymerase chain reaction with primers designed to hybridise to the known DNA sequence flanking exon 3
  • the forward pnmer was 5'- TACTCCCGAGTCTCCGGGTCTG-3 * (SEQ ID NO 1) and the reverse pnmer was 5'- GAGGCGCCCCACTGCCCCTGGT-3'
  • the polymerase chain reaction was carried out in a total volume of 25 ⁇ l, with lOOng genomic DNA, 50ng of each primer. 0 2mM of each deoxynucleoside triphosphate (dATP. dCTP. dGTP and dTTP) 50mM KC1, lOmM T ⁇ s-HCl, pH 9 0 at 25°C, 0 1% Triton X-100, 1 5mM MgCl and 0 5U of Taq Polymerase Reaction mixtures were covered with an equal volume of mineral oil and amplification was carried out using the "hot start" technique in a thermal cycler The conditions for amplification involved denaturation at 94 °C for 5 min followed by addition of Taq Polymerase Thirty cycles were then performed 94 °C for 1 min. 63°C for 1 mm. 72°C for 1 mm and finally a 10 mm extension at 72°C
  • Genotyping of the C/T-93 HLA-G polymo ⁇ hism was then performed using a semi nested amplification approach and the Glycosylase Mediated Polymo ⁇ hism Detection method (Vaughan and McCarthy 1998)
  • a 319bp section of the HLA-G gene encompassing the C/T-93 polymo ⁇ hism location was amplified using a semi nested polymerase chain reaction approach from the previously amplified exon 3 of the HLA-G gene using the exon 3 reverse primer and the internal fonvard primer 5'- GACCGAGGGGGTGGGGCCAGGTTCT-3 * (SEQ ID NO 3)
  • the forward primer was end labelled by incubation with polynucleotide kinase in the manufacturers buffer (New England Biolabs) and 5 ⁇ C ⁇ 32P- ATP (3000C ⁇ /mmol) for 30 min at 37°C followed by ethanol precipitation to remove unused labelled nucleotide
  • dCTP, dGTP and dUTP 50mM KC1, lOmM T ⁇ s- HC1 pH 9 0 at 25°C 0 1% Triton X- 100,1 5mM MgCl and 0 5U of Taq Polymerase Reaction mixtures were covered with an equal volume of mineral oil and amplification was carried out using the "hot start" technique m a thermal cycler The conditions for amplification involved denaturation at 94°C for 5 min followed bv addition of Taq Polymerase Thirty cycles were then performed 94°C for 1 mm. 64°C for 1 mm.
  • reaction mixture was then treated with exonuclease I to digest the primers not extended m the amplification step This was achieved by incubating the PCR reaction mixture with 0 4 units of exonuclease I at 37°C for 30 min The exonuclease was subsequently heat inactivated by incubating the reaction at 80°C for 15 min
  • PCR amplification was carried out in 25 ml reactions, each of which contained 100 ng genomic DNA PCR buffer (100 mMT ⁇ s- HC1 pH 8 3 (20°C), 500 mM KC1, 15 mM MgCl 2; ), 200 mM of each dNTP, 300 nM of each primer and
  • the common 14 base pair insertion / deletion polymo ⁇ hism in exon 8 of the HLA-G gene is referred to as I/D-E8 herein (also known as where I-E8 is one allele of the polymo ⁇ hism and D-E8 is the other allele of the polymo ⁇ hism
  • Genotyping of the HLA-G exon 8 deletion polymo ⁇ hism was performed by amplifying a short section flanking the deletion location m exon 8 This was achieved using the polymerase chain reaction with primers designed to hybridise to known DNA sequence in exon 8
  • the forward primer was 5'-TGTGAAACAGCTGCCCTGTGT-3' (SEQ ID NO 10) and the reverse primer was S'-AAGGAATGCAGTTCAGCATGA ⁇ ' (SEQ ID NO 11)
  • the polymerase chain reaction was carried out in a total volume of 25 ⁇ l, with lOOng genomic DNA, 50ng of each primer, 0 2mM of each deoxy ⁇ bonucleoside triphosphate (dATP, dCTP, dGTP and dTTP) 50mM KC1. lOmM T ⁇ s-HCl. pH 9 0 at 25°C. 0 1% Triton X-100. 0 5mM MgCl and 0 5U of Taq
  • Polymerase Reaction mixtures were covered with an equal volume of mineral oil and amplification was carried out using the "hot start" technique in a thermal cycler
  • the conditions for amplification involved denaturation at 94°C for 5 mm followed by addition of Taq Polymerase Thirty cycles were then performed 94°C for 1 mm.
  • the I/D exon 8 polymo ⁇ hism was genotyped by size separation of the PCR products on a 10% non denaturing polyacrylamide gel and visualised by staining with ethidium bromide, the I-E8 insertion allele giving rise to a 151 bp product (SEQ ID NO 12) and the D-E8 deletion allele giving rise to a 137 bp product (SEQ ID NO 13)
  • Allele specific genotyping In order to gain more information from the transmission of HLA-G polymo ⁇ hisms in the second phase of the work, allele specific genotyping was performed In the majority of cases maternal and paternal C/T-93 and I/D-E8 HLA-G haplotypes could be directly assigned In cases where all members of a trio were heterozygous for either C/T-93 or I/D-E8 polymo ⁇ hisms, allele specific amplification was performed in order to assign haplotypes This was achieved using allele specific primers which allowed selective amplification of the I-E8 or D-E8 allele Following allele specific amplification, the C/T-93 polymo ⁇ hism was then genotyped using the GMPD assay described above Conditions for amplification of the I-E8 allele were 30 cycles at 94°C for 45s, 64°C for 45s.
  • the frequencies of the C-93 and T-93 allele in the pre-eclamptic offspring were 0 537 and 0 463 respectively In comparison, the frequencies in the control offspring were 0 604 and 0 396 respectively
  • the expected distribution of genotypes should be C-93/C-93 - 0 36.
  • C-93/T-93 0 478.
  • T-93/T-93 0 156
  • the distribution of genotypes in the pre-eclamptic offspnng group is significantly different (Chi-square - 11 01, p ⁇ 0 001, table 1)
  • normal allele/deletion allele 0 497.
  • deletion allele/deletion allele 0 212
  • normal allele/deletion allele 0 500.
  • deletion allele/deletion allele 0 241
  • the parents of the pre-eclamptic and control offspring were genotyped for the HLA-G C/T-93 genotype and the genotypes were analysed in conjunction with the offspring genotypes In this analysis we scored the number of cases where the offspring had inherited a paternal C/T-93 allele that was not present m the maternal genotype For the pre-eclamptic offspring. 41 % of cases had a paternal allele of the C/T-93 genotype that was not present in the maternal genotype By contrast, for the control offspnng. 28% of cases had a paternal C/T-93 allele not represented in the maternal genotype
  • the transmission disequilibrium test transmission disequilibrium test assesses whether assess whether transmission of maternal and paternal alleles from heterozygous parents to offspring differed from the null expectation (of 50 50) and is valid even when Hardy- Weinberg equilibrium is violated by unusual population structure
  • the transmission disequilibrium test was applied, the transmission of the C/T- 93 and I/D-E8 alleles to offspring did not differ from the null expectation (Table 5)
  • significant 26 deviations from the null expectation were observed when maternal and paternal transmission frequencies were analysed independently of each other
  • the p ⁇ magravida mothers investigated here differed significantly from Hardy-Wemberg expectations (pj 0 006) for I/D-E8 genotype frequencies (observed genotype frequencies I/I, 17. I/D. 58. D/D. 15, expected I/I. 24, I/D. 45. D/D. 21)
  • a and E 11 of the 14bp of the polymo ⁇ hism is repeated in intron seven of the HLA-G gene
  • the core sequence "atttgt " ' is repeated one or more times m the 3 " UTR of all class I genes but is absent in coding sequences
  • Examination of the secondary structure around the I/D-E8 polymo ⁇ hism using the mfold programme (Zuker. 1994) shows that the 14n sequence is involved in a region of the 3'UTR having extensive secondary stmcture and that the secondary structure is altered depending on the presence or absence of the 14n sequence (data not shown)
  • the presence or absence of the polymo ⁇ hism may affect the stability and/or alternative splicing of HLA-G mRNA through formation of alternative secondary structures
  • HLA-G genotypes and haplotypes in pre-eclampsia trios were examined independently using transmission segregation analysis pre-eclampsia trios were also compared to the cohort of control p ⁇ magravida trios
  • a significant difference was also observed for the allele frequency of the I/D-E8 polymo ⁇ hism between control and pre-eclampsia fathers (p j 0 02.
  • the genotypes and haplotypes of the couples are shown in Table 16
  • the 93-E8 haplotypes of female and male mating partners were constructed 50% of couples have the possibility of producing foetuses with the maternally transmitted C-93/D-E8 haplotype and the paternally transmitted T-93/I-E8 haplotype combination This compares with a 24% possibility in control couples and a 44% possibility in pre-eclampsia couples
  • the possibility of producing foetuses with the maternally transmitted D-E8 allele and the paternally transmitted I-E8 allele is 46% for control couples, 70% for pre-eclampsia couples and 85% for recurrent miscarriage couples
  • Maternally transmitted C-93/D-E8 haplotype and the paternally transmitted T-93/I-E8 haplotype combinations in foetuses are only found in pre-eclampsia offspring Recurrent miscarriage couple no 15 must produce such a foetus
  • Table 17 also had the same haplotype combinations This provides evidence that partners where the female is homozygous for the T-93/I-E8 haplotype and the male has the C-93/D-E8 and T-93/I-E8 haplotypes are susceptible to pre-eclampsia and/or miscarriage Taken together, the results 32 provide evidence that miscarriage and pre-eclampsia are closely related and that miscarriage is a severe expression of PE
  • One offspring of T-93/I-E8 and C-93/D-E8 haplotype combination in control trios was found where the mother was homozygous for T-93/I-E8 and father had the C-93/D-E8 haplotype and the C-93/I-E8 haplotype
  • the maternally transmitted C-93/D-E8 haplotype and the paternally transmitted T-93/I-E8 haplotype combination cause pre-eclampsia and miscarriage in primagravidas
  • There were five second offspring bearing the maternally transmitted C-93/D-E8 haplotype and the paternally transmitted T-93/I-E8 haplotype combination (Table 18)
  • HLA-G is a susceptibility gene for normal pregnancy, pre-eclampsia and miscarriage As pre-eclampsia is associated with intra uterine growth retardation, the HLA-G gene is also a susceptibility gene for intra uterine growth retardation As miscarriage frequently occurs so early that it is not detected, the HLA-G is also a susceptibility gene for miscarriage related unexplained infertility Exposure to foetal antigens including HLA-G in the first pregnancy has been shown to induce tolerance to antigens that are problematic in first pregnancy and thus provide a means for potential treatment of pre- eclampsia. miscarriage, intrauterine growth retardation, miscamage related infertility and autoimmune disease and provide a means to induce tolerance to foreign antigens for pu ⁇ oses such as transplantation of foreign tissue
  • HLA-G I/D-E8 polymo ⁇ hism has been investigated previously in pre-eclampsia and no detectable relationship was observed between susceptibility to pre-eclampsia and HLA-G (24) This result is consistent with the results reported here in that an HLA-G effect is not seen when the I/D-E8 polymo ⁇ hism is independently analysed by association studies alone
  • results presented here show that genetic screening of maternal and/or paternal and/or foetal DNA is of value for predictive testing of susceptibility to pre-eclampsia, eclampsia, intrauterine growth retardation, miscarriage and miscarriage-related infertility Furthermore, transmission of HLA-G alleles to offspring in normal pregnancv differs from the normal expectation Therefore, the results presented here show that genetic screening of maternal and/or paternal foetal DNA is of value for predictive testing of susceptibility to normal pregnancy 34
  • foetal nucleic acid is isolated from any material containing nucleic acid of foetal origin in the mother such as amniotic fluid, maternal blood or cho ⁇ onic villus
  • the results show that genetic screening of parents will also be of value for predictive testing of susceptibility to pre-eclampsia
  • HLA-G is an excellent candidate for pre-eclampsia since HLA-G is considered to play a key role m foetal-matemal immune interactions
  • the C/T-93 HLA- G allele associated with pre-eclampsia is a silent polymo ⁇ hism but its effect on HLA-G mRNA stability or splicing is unknown It is likely that this polymo ⁇ hism and/or variations linked to this polymo ⁇ hism play a causative role in pre-eclampsia
  • HLA-G is now known to induce synthesis IL-3 and IL-1 beta and down-regulate tumour necrosis factor-alpha production
  • HLA-G primary transcript is alternatively spliced to yield several 35 different mRNAs
  • One of these alternatively spliced forms includes intron 4
  • the open reading frame in this mRNA continues into intron 4 terminating 21 amino acids after the alpha3 domain - encoded by exon 4
  • the transmembrane region encoded by exon 5 and the cytoplasmic tail of HLA-G is excluded
  • the resultant protein is hence soluble
  • the HLA-G genotypes associated with pre-eclampsia may lead to variations in HLA-G mRNA and/or HLA-G protein which in turn could be detected by characterisation of HLA-G mRNAand or protein
  • characterisation of HLA-G protein in pregnant females, foetuses and/or respective male mating partner would allow one to diagnose susceptibility to pre-eclampsia and miscarriage
  • expression of the HLA-G protein leads directly or indirectly to alterations in the levels of certain molecules such as IL-3, IL-1 beta and/or tumour necrosis factor alpha
  • the HLA-G genotypes associated with pre-eclampsia may result in changed expression of such molecules
  • measuring of the levels of such molecules and comparing these levels with the normal observed levels would allow one to diagnose susceptibility to pre-eclampsia and miscarriage
  • HLA-G genotypes associated with pre-eclampsia may result in decreased expression of HLA-G This in turn would lead to increased lysis of trophoblasts by NK cells
  • measuring of the levels of trophoblast specific marker and comparing these levels with the normal observed levels would allow one to diagnose susceptibility to pre-eclampsia and miscarriage
  • HLA-G variants associated with pre-eclampsia and miscarriage and normal pregnancy are likely to have one of a small number of consequences l) a variant could result in altered expression of HLA-G splice forms and levels thereof which would be reflected as altered levels of HLA-G splice forms including soluble HLA-G in the serum
  • measuring of size, levels and/or splice forms of HLA-G mRNA and/or protein including soluble HLA-G levels and comparing these levels with the normal observed levels would allow one to diagnose susceptibility to pre-eclampsia and miscarriage
  • the HLA-G variants associated with pre-eclampsia and miscarriage may result in va ⁇ ations in HLA- G protein which in turn could be detected by protein characterisation of cellular and/or soluble HLA-G
  • characterisation of HLA-G protein in pregnant females, foetuses and/or respective male mating partner would allow one to diagnose susceptibility to pre-eclam
  • the HLA-G variants associated with pre-eclampsia and miscarriage may result in increased or decreased expression of paternal and/or maternal HLA-G This in turn would lead to increased lysis of trophoblasts by NK cells and/or cytotoxic T cells
  • measuring of the levels of trophoblast specific marker and comparing these levels with the normal observed levels would allow one to diagnose susceptibility to pre-eclampsia
  • the HLA-G variants associated with pre-eclampsia and miscarriage may result in increased or decreased cell cleavage rates in the embryo
  • measuring of cell cleavage rates in cells expressing one or more HLA-G variants and any combinations thereof would allow one to diagnose susceptibility to pre- eclampsia and miscarriage
  • the results show that HLA-G polymo ⁇ hism plays a major role in predisposition to normal, pre- eclampsia and miscamage outcome in pregnancy and that haplotypic combinations and parent-of
  • a protective foetal-maternal HLA-G allele is likely to arise through the transmission of a dominant maternal allele to the foetus which is recognised as self by the maternal immune system
  • a protective foetal-paternal allele is likely to arise through cross recognition of the paternal allele as self by the maternal immune system
  • a problematic foetal-paternal allele is likely to arise through cross recognition of the paternal allele as non- self by the maternal immune system
  • the results indicate maternal education of the lymphocyte repertoire for maternal HLA-G during and/or prior to pregnancy and for paternal HLA-G during pregnancy
  • the results also indicate certain paternal HLA-G alleles are compatible with the maternal immune system while others are less compatible Combinations of less compatible/incompatible paternal HLA-G alleles with maternal alleles which do not protect against the paternal alleles are likely to cause susceptibility to pre-eclampsia and miscarriage
  • HLA-G is capable of protecting otherwise susceptible target cells from natural killer cell mediated lysis through its interaction with inhibitory receptors on natural killer cells
  • HLA-G is also capable of stimulating an HLA-G restricted lymphocyte response.
  • HLA-G molecules can serve as target molecules in lytic reactions with lymphocytes, and HLA-G is involved in education of the lymphocytic repertoire
  • pre-eclampsia, miscarriage, miscarriage-related infertility and intrauterine growth retardation is likely to arise through a mechanism involving blood mononuclear cells such as natural killer cells and cytotoxic T lymphocytes whereby interaction between the female mating partner ' s T cells and foetal antigens is compromised by comparison with normal pregnancy
  • compromised interaction leading to the lack of tolerance leads to cell killing
  • Compromised interaction also can lead to lack of stimulation of cells expressing HLA-G molecules and/or lack of stimulation of cells interacting with cells expressing HLA-G molecules
  • miscarriage, miscarriage-related infertility and mtra-uterine growth retardation arises from lack of education and/or inadequate induction of education to the foetal antigens in the female mating partner during and/or prior to pregnancy
  • Lack of and/or compromised induction of education to paternal antigens such as HLA-G in the foetus and/or a defective HLA-G interaction with natural killer cells could lead to lysis of trophoblasts and/or lack of stimulation of trophoblasts leading to reduced trophoblast function and/or lack of stimulation of cells interacting with trophoblasts
  • HLA-G linked conditions such as pre-eclampsia.
  • miscarriage, miscarriage-related infertility and intrauterine growth retardation are likely to arise through blood mononuclear cell mediated killing of accessible foetal tissues such as trophoblasts and/or lack of stimulation of trophoblastic cells because of compromised HLA-G interaction with blood mononuclear cells trophoblasts and/or lack of stimulation of blood mononuclear cells because of compromised HLA-G interaction with trophoblastic cells
  • MHC major histocompatibility
  • diagnosis of susceptibility to pre-eclampsia. miscarriage, miscarriage-related infertility and intrauterine growth retardation and prediction of pregnancy outcomes may be achieved by direct and indirect measurement of the education in the female mating partner to foetal antigens and/or direct and indirect measurement of the interaction between blood mononuclear cells and HLA-G and/or HLA-G expressing cells
  • direct and indirect measurement of the education in the female mating partner to foetal antigens and/or direct and indirect measurement of the natural killer cell activity in the female mating partner to HLA-G expressing cells and/or direct and indirect measurement of the interaction between blood mononuclear cells and HLA-G and/or HLA-G expressing cells offers a means to monitor the course of pregnancy
  • Induction of education to foetal antigens in the female mating partner by treatment with HLA-G and/or peptides known to bind to HLA-G constitutes a therapeutic means for prevention and/or treatment of pre- eclampsia, miscarriage, miscarriage-related infertility and intrauterine growth retardation and any other HLA-G related disorders
  • inhibition of the interaction of one or more HLA-G variants with blood mononuclear cells may be achieved by inactivating the HLA-G gene or HLA-G gene variant and/or one or more HLA-G receptors on blood mononuclear cells
  • This may be achieved through the use of one or more gene 40 inactivating approaches such as treatment with one or more nucleic acid antisense and/or ribozyme molecules which inhibit expression of the HLA-G gene and/or HLA-G gene vanant and/or one or more HLA-G receptors on blood mononuclear cells
  • This may be also be achieved by inactivating the HLA-G gene in one or both partners of a mating couple somatically or in the germ line through the use of gene therapy approaches whereby inhibitory nucleic acid based molecules such as antisense, and/or ribozyme are introduced into an individual
  • This may be also be achieved by inactivating the HLA-G gene in one or both partners of a mating couple somatically or in the germ
  • HLA-G binds a diverse but limited array of peptides m a manner similar to that found for classical class I molecules and it has been reported that HLA-G is expressed in the human thymus raising the possibility that maternal unresponsiveness to HLA-G expressing foetal tissues may be shaped in the thymus by central presentation of this MHC molecule on the medullary epithelium (C ⁇ sa et al 1997) HLA-G is known to be capable of stimulating a HLA-G restricted cytotoxic T lymphocyte response and HLA-G molecules can serve as target molecules in lytic reaction with cytotoxic T lymphocytes and HLA-G expressed internally in vivo in transgenic animals is involved in education of the lymphocytic repertoire (Schmidt et al .
  • the invention shows that the induction of education to foetal antigens occurs during pregnancy and arises from exposure of the mother to foetal antigens during pregnancy HLA-G allele combinations that were unacceptable in first pregnancy and/or were linked to pre-eclampsia were acceptable in second pregnancy without any associated pregnancy complications
  • induction of education to foetal antigens is likely to arise from a process involving HLA-G
  • the invention offers a means of inducing education including tolerance to HLA-G and/or peptides bound to HLA-G in an individual through mimicking the exposure to foetal antigens that occurs during pregnancv
  • treatment of an individual with HLA-G and or / peptides known to bind to HLA-G constitutes a means to 41 induce education in an individual to antigens
  • this offers a means to induce tolerance to antigens that cause susceptibility to pre-eclampsia, susceptibility to miscarriage, autoimmune disease and transplant rejection
  • miscarriage, miscarriage- related infertility and intrauterine growth retardation and any other HLA-G related disorders the alteration of the level and/or activity of molecules arising from the interaction of HLA-G and/or HLA-G expressing foetal cells with blood mononuclear cells such as lymphocytes and natural killer cells is likely to be compromised by comparison with that occurring during normal pregnancy
  • mimicry of the alteration of the level and or activity of one or more molecules arising from the interaction of HLA-G and/or HLA-G expressing foetal cells with blood mononuclear cells in an individual constitutes a therapeutic means for prevention and/or treatment of pre-eclampsia.
  • miscarriage, miscarriage-related infertility and intrauterine growth retardation and any other HLA-G related disorders the alteration of the level and/or activity of molecules arising from the interaction of HLA-G and/or HLA-G expressing foetal cells with blood mononuclear cells in an individual constitutes a
  • the deficit of maternal HLA-G C-93/D-E8 genotypes and the excess of T-93/I-E8 genotypes transmitted to control offspring but not to pre-eclampsia offspring implies selection of foetuses in normal pregnancy dependent on HLA-G genotype
  • selection of one or both mating partners, sperm and/or egg donors and/or embryo recipients based on male and/or female HLA-G and/or HLA-G homologue genotypes and/or serotypes and/or activity associated with a successful normal first pregnancy with a specific mating partner offers a means to improve fertility and the success rate of m vitro fertilisation and embrvo transfer in animals and improve pregnancy outcome
  • HLA-G protects trophoblasts from blood mononuclear cell mediated killing
  • direct and indirect measurement of measurable substances which originate from trophoblast cell killing should allow diagnosis of susceptibility to pre-eclampsia, miscarriage, intra-uterine growth retardation . and monitoring of pregnancv for normal progress, and progress towards pre-eclampsia.
  • miscarriage and mtra-uterine growth retardation in humans and animals More specifically, the interaction between MHC molecules such as HLA-G and blood mononuclear cells is known to directly and indirectly alter the synthesis and levels of several cytokines Similarly, trophoblasts are known to synthesise and secrete several cytokines In particular, the altered regulation of some of these cytokines would be expected to compromise the foetal - maternal immune interaction and could be manifest as pre-eclampsia and/or eclampsia and/or intrauterine growth retardation and/or miscarriage and/or miscarriage-related infertility For example, the interaction of HLA-G expressing cells with blood mononuclear cells increases the amount of mterleukin-
  • Trophoblasts are known to produce the lmmunosuppressive cytokine interleukin 10 - a cytokine that potently inhibits alloresponses in mixed lymphocyte reactions
  • Trophoblasts are also known to produce interleukin 2.
  • a cytokine that both protects the foetus and in involved in activation of maternal killer cells to protect against invading trophoblasts interleukin 4 and its receptor, which play a role in regulation of umbilical blood flow mediated through the induction of cyclooxygenase-2.
  • interleukin 6 which is likely to play a role in tissue remodelling associated with placentation
  • HLA-G function and HLA-G expression can be measured
  • screening for agents which alter the expression and/or function and/or which mimic the function of HLA-G provide a method for screening for potential pre-eclampsia. miscarriage, miscarriage-related infertility and intrauterine growth retardation therapeutic agents
  • HLA-G human leukocyte antigen
  • HLA-G bind identical sets of endogenous peptides but differ with respect to TAP association Immunity, 3, 591-600 (1995)
  • Table 1 Genotype and allele distribution of the HLA-G C/T-93 (C1488D and I D-E8 (exon deletion) polvmo ⁇ hisms in pre-eclamptic and control ofFsrping.
  • Table 2 Genotype and allele distribution of the HLA-G C/T-93 and I D-E8 polymo ⁇ hisms in primigravida trios.
  • Offspring 90 24(26.7) 41(45.5) 25(27.8) 0.49/0.51
  • the model was reduced by stepwise elimination of parameters whose significance was greater than 0.10.
  • Probability values (p) are presented with the numbers of degrees of freedom as a subscript.
  • MT/MNT/PT/PNT maternally transmitted / maternally non- transmitted / paternally transmitted / paternally non-transmitted.
  • Table 7 Transmitted and non-transmitted HLA-G haplotypes to offspring in primigravida trios.
  • Table 8 Transmitted and non-transmitted HLA-G haplotypes in trios.
  • Haplotype Offspring Haplotype Mothers Fathers n MT - PT n T - NT n
  • T Haplotype transmitted to offspring
  • NT Haplotype non-transmitted to offspring
  • MT Haplotype transmitted from mother to offspring
  • PT Haplotype transmitted from father to offspring
  • Table 9 Genotype and allele distribution of the HLA-G C T-93 and I D-E8 polymorphisms in pre-eclampsia primigravida trios.
  • Offspring 90 24(26.7) 41(45.5) 25(27.8) 0.49/0.51 21(23.3) 47(52.2) 22(24.5) 0.49/0.51
  • Offspring 82 18(22.0) 57(69.5) 7(8.5) 0.57/0.43 14(17.1) 55(67.1) 13(15.8) 0.51/0.49 51
  • Table 10 Transmitted and non-transmitted HLA-G haplotypes to offspring in control and pre-eclampsia trios.
  • Table 11 Genotype mating outcomes for the HLA-G polymo ⁇ hisms in control and pre- eclampsia trios.
  • Control Control PE PE Offspring Mothers Fathers Offspring Mother Fathers s
  • MT maternally transmitted
  • MNT maternally non-transmitted
  • Probability values (p) are presented with the numbers of degrees of freedom as a subscript.
  • Probability values (p) are presented with the numbers of degrees of freedom as a subscript.
  • Table 14 Transmitted and non-transmitted HLA-G haplotypes in control and pre- eclampsia trios.
  • T-D T-D 4 0 T-D T-D 2 3 1 0
  • T-I T-I 11 6 T-I T-I 9 5 8 7 n 84 63 84 84 70 68
  • MT haplotype transmitted from mother to offspring
  • PT haplotype transmitted from father to offspring
  • T haplotype transmitted to offspring
  • NT haplotype non-transmitted to offspring
  • Table 15 Relative risk of foetal, maternal and parent of origin effects in a log linear model
  • Table 17 HLA-G haplotypes in recurrent miscarriage couples.
  • Table 18 Transmitted and non-transmitted HLA-G haplotypes (extended genotypes) in first and second offspring of normal mothers
  • MT haplotype transmitted from mother to offspring
  • PT haplotype transmitted from father to offspring
  • T haplotype transmitted to offspring
  • NT haplotype non-transmitted to offspring
  • Table 19 Transmitted and non-transmitted HLA-G extended genotypes in first and second offspring of primigravida pre-eclampsia mothers
  • MT haplotype transmitted from mother to offspring
  • PT haplotype transmitted from father to offspring

Abstract

L'invention porte sur l'identification d'un gène de susceptibilité aux pré-éclampsies et éclampsies et sur un procédé et une trousse pour diagnostiquer la susceptibilité à une grossesse normale, à la pré-éclampsie, à l'éclampsie, au retard de croissance intra-utérin, aux fausses-couches, ou à la stérilité due aux fausses-couches. L'invention se base sur l'analyse du HLA-G, ou de l'acide nucléique lié au HLA-G, ou de la protéine HLA-G, ou de l'ARNm du HLA-G, ou de cellules ou molécules dont la concentration varie sous l'action du HLA-G. L'invention porte également sur des préparations pharmaceutiques et des méthodes traitant les états précités.
PCT/IE1999/000012 1998-02-25 1999-02-25 Gene de susceptibilite a la pre-eclampsie et aux fausses-couches lie au hla WO1999043851A1 (fr)

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AU26363/99A AU2636399A (en) 1998-02-25 1999-02-25 Hla linked pre-eclampsia and miscarriage susceptibility gene
CA002321223A CA2321223A1 (fr) 1998-02-25 1999-02-25 Gene de susceptibilite a la pre-eclampsie et aux fausses-couches lie au hla
EP99906418A EP1056886A1 (fr) 1998-02-25 1999-02-25 Gene de susceptibilite a la pre-eclampsie et aux fausses-couches lie au hla
NO20004222A NO20004222L (no) 1998-02-25 2000-08-23 HLA-koblet pre-eklampsi- og abort-mottagelighetsgen
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DE19952509A1 (de) * 1999-11-03 2001-05-31 Grosse Wilde Hans Nachweis und Verwendung löslicher HLA-G Moleküle
FR2810047A1 (fr) * 2000-06-13 2001-12-14 Commissariat Energie Atomique Nouvelle isoforme d'hla-g et ses applications
WO2005108624A2 (fr) * 2004-05-06 2005-11-17 University Of Chicago, Uctech Utilisation du genotypage hla-g dans des affections d'origine immunologiques
WO2006069592A2 (fr) * 2004-12-31 2006-07-06 Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patientenzorg Methode permettant de diagnostiquer et/ou de predire la toxemie preeclamptique et/ou des troubles associes
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WO2008000089A1 (fr) * 2006-06-30 2008-01-03 Librach Clifford L Procédé de détection d'une pré-éclampsie
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EP2110442A1 (fr) * 1999-09-08 2009-10-21 Genzyme Corporation Procédés pour la détection de maladies
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US9109256B2 (en) 2004-10-27 2015-08-18 Esoterix Genetic Laboratories, Llc Method for monitoring disease progression or recurrence
US9777314B2 (en) 2005-04-21 2017-10-03 Esoterix Genetic Laboratories, Llc Analysis of heterogeneous nucleic acid samples
EP3648110A1 (fr) * 2018-11-02 2020-05-06 Immune Compass Ltd. Système de criblage d'un trouble lié au système immunitaire
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DE19952509A1 (de) * 1999-11-03 2001-05-31 Grosse Wilde Hans Nachweis und Verwendung löslicher HLA-G Moleküle
FR2810047A1 (fr) * 2000-06-13 2001-12-14 Commissariat Energie Atomique Nouvelle isoforme d'hla-g et ses applications
WO2001096564A3 (fr) * 2000-06-13 2002-03-14 Commissariat Energie Atomique Isoforme d'hla-g (hla-g7) et ses applications
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JP2010122238A (ja) * 2003-03-25 2010-06-03 Proteogenix Inc 生体液のプロテオーム分析
WO2005108624A2 (fr) * 2004-05-06 2005-11-17 University Of Chicago, Uctech Utilisation du genotypage hla-g dans des affections d'origine immunologiques
WO2005108624A3 (fr) * 2004-05-06 2006-05-04 Univ Chicago Uctech Utilisation du genotypage hla-g dans des affections d'origine immunologiques
US9109256B2 (en) 2004-10-27 2015-08-18 Esoterix Genetic Laboratories, Llc Method for monitoring disease progression or recurrence
WO2006069592A3 (fr) * 2004-12-31 2008-01-03 Vereniging Voor Christelijk Hoger Onderwijs Wetenschappelijk Onderzoek En Patientenzorg Methode permettant de diagnostiquer et/ou de predire la toxemie preeclamptique et/ou des troubles associes
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WO2007087539A2 (fr) * 2006-01-24 2007-08-02 The University Of Chicago Site de liaison de polymorphisme nucléotidique simple (snp) pour des micro arn dans l'antigène hla-g
WO2007087539A3 (fr) * 2006-01-24 2007-09-13 Univ Chicago Site de liaison de polymorphisme nucléotidique simple (snp) pour des micro arn dans l'antigène hla-g
WO2008000089A1 (fr) * 2006-06-30 2008-01-03 Librach Clifford L Procédé de détection d'une pré-éclampsie
US8673564B2 (en) 2007-06-07 2014-03-18 Haplomic Technologies Pty Ltd In situ methods for gene mapping and haplotyping
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