JP2003517267A - HLA-linked preeclampsia and miscarriage susceptibility genes - Google Patents

Abstract

  (57) [Summary] The present invention relates to the identification of susceptibility genes for preeclampsia and eclampsia, and methods and diagnostic kits for diagnosing susceptibility to normal pregnancy, preeclampsia, eclampsia, intrauterine growth retardation, miscarriage or miscarriage-related infertility I will provide a. The present invention is based on the analysis of HLA-G or HLA-G linked nucleic acids, or HLA-G proteins or HLA-G mRNA or HLA-G cells or HLA-G molecules whose concentration changes as a result of HLA-G action. The present invention also provides pharmaceutical compositions and methods for treating the above conditions.

Description

Detailed Description of the Invention

The present invention relates to susceptibility genes for pre-eclampsia and eclampsia and the use of such genes in methods for diagnosing susceptibility to these diseases. The invention also relates to test kits for the diagnosis of susceptibility and pharmaceutical compositions for the prevention or treatment of this disease. The present invention may also be used in the diagnosis of miscarriage and / or abortion-related infertility and / or susceptibility to intrauterine growth retardation.

[0002] In particular, the present invention provides a predisposition gene for HLA-linked human preeclampsia and miscarriage (H
LA-G), some alleles, or linked alleles of linked genes that cause susceptibility to preeclampsia and abortion. More particularly, the invention relates to sequence changes in the HLA-G and linked genes and their use in diagnosing susceptibility to preeclampsia and miscarriage. The invention further provides for sequence changes in the HLA-G gene,
And their use in the diagnosis of preeclampsia and miscarriage. Furthermore, the invention relates to treatments for pre-eclampsia and abortion and susceptibility to pre-eclampsia and abortion. Such treatments include protein therapy, gene therapy, and protein mimetics. The invention also relates to screening for drugs for the treatment of preeclampsia and miscarriage, and for the treatment of susceptibility to preeclampsia and miscarriage. Finally, the invention relates to the screening of HLA-G and HLA-G linked genes for sequence alterations useful for diagnosing susceptibility to preeclampsia and miscarriage.

Preeclampsia is a major cause of fetal and maternal morbidity and mortality, with possibly long-term adverse effects on health due to long-term associated intrauterine hypoxia. Pre-eclampsia occurs in approximately 5-10% of the total birth population and is a unique pregnancy disease. Acute pathological changes begin to change shortly after delivery. The pathological mechanisms that cause preeclampsia are unclear and no markers have been identified that can predict the disease prior to clinical evidence of the disease. Furthermore, an association has been observed between abortion and preeclampsia (Cooper et al., 1988).

Epidemiological studies indicate that the disease is highly inherited, predominantly bound to the first pregnancy, and largely prevented by a normal first pregnancy with the same partner. Patients affected by the first pregnancy have a 13.1% recurrence risk in the second pregnancy, whereas in the normal first pregnancy, the onset in the second pregnancy is of the order of 1%. Therefore, the first pregnancy appears to have a significant protective effect against preeclampsia in subsequent pregnancies. Therefore, it is understood that preeclampsia is generally avoided (Lie et al., 1998).

Several classification schemes have been proposed to help the clinical perception of preeclampsia. US National Institute on Hypertension in Pregnancy
The categories claimed by the es of Health Working Group are:
> 15 mmHg diastolic blood pressure or> 30 mmH from early pregnancy measurements
increased systolic blood pressure of g or> 140/90 mmHg in late pregnancy (if no early reading is possible); + proteinuria (> 0.3 g / 24 h) and / or edema (odema) . However, in practice proteinuria measurements may not always be determined. Symptoms in addition to elevated blood pressure (eg, headache, visual impairment, and / or epigastric pain) indicate worsening in pregnancy consistent with preeclampsia,
And form the basis for clinical intervention of early delivery by Caesarean section to resolve the condition. Spinillo et al. (1994) reported that women with pre-eclampsia had gestational age (SG
A) reported that the infant had a significantly increased incidence of small intrauterine growth retardation (IUGR).

Although the cause of preeclampsia is unknown, hypertension has been observed in preeclampsia and has been the focus of extensive research into the disorder. However, the pathological and physiological changes in preeclampsia indicate that this syndrome is much more common than pregnancy-induced hypertension. Evidence to date indicates the action of the placental trophoblast as the underlying cause.

In preeclampsia, the trophoblast cell layer invasion is shallow and the spiral arterial invasion is aberrant, resulting in diminished villous interstitial blood perfusion. Furthermore, the characteristic pattern of integrin conversion that occurs during normal trophoblast differentiation does not occur in preeclampsia.

The outermost layer (trophoblast) of the human placenta is the classical class I human leukocyte antigen (HLA-).
A and HLA-B) and class II proteins (HLA-DR, HLA-D
Q, and HLA-DP). This prevents recognition by maternal T lymphocytes, but the lack of class I molecules makes these cells susceptible to attack by natural killer (NK) cells. However, trophoblasts in direct contact with maternal tissue preferentially express characteristic nonclassical class Ib molecules. HLA-G, HLA-E,
And limited expression of HLA-C also occurs. Expression of HLA-G has been shown to be sufficient to protect otherwise sensitive target cells from NK cell-mediated lysis. NK cells usually simultaneously express several different inhibitory receptors of varying specificity. Cross-linking of any one inhibitory receptor is sufficient to inactivate NK cell activity against all possible targets. Membrane-bound HLA-G
The molecules are NK inhibitory receptor 1 and inhibitory receptor 2 (NK1 and NK
It has been shown that 2) can be used to inhibit autoreactive NK cells. CD94 /
NKG2 has been shown to be the predominant inhibitory receptor involved in the recognition of HLA-G by decidual cells and peripheral NK cells. Thus, at a functional level, HLA-G may protect target cells from destruction by NK-1, NK-2, and NKG2-specific effector cells (Loke and King, 1997).
More recently, HLA-G has been shown to regulate the ability of blood mononuclear cells to release cytokines (Maejima et al., 1997), suggesting a role for HLA-G in causing maternal immune reciprocal crossing. To do. Specifically, peripheral blood mononuclear cells (
Co-culture of HLA-G expressing cells with PBMC) resulted in interleukin-3 (IL
-3) and interleukin-1β (IL-1β), and PB
It reduced the amount of tumor necrosis factor alpha (TNF-alpha) release from MC cells.

HLA-G binds to a diverse but limited peptide array in a manner similar to that found for classical class I molecules, and HLA-G is expressed in human thymus and HLA-G is expressed. Maternal non-response to G-expressing fetal tissue has been reported to increase the likelihood that it can be embodied in the thymus by central presentation of this MHC molecule on spinal epithelium (Crisa et al., 1997). HLA-G is HLA-
It is known to be able to stimulate a G-restricted cytotoxic T lymphocyte response, and HL
The AG molecule can serve as a target molecule in the lytic reaction with cytotoxic T lymphocytes and is expressed in vivo in transgenic animals in vivo HL.
AG is involved in the education of the lymphocyte repertoire (Schmidt et al., 1997.
).

Major histocompatibility (MHC) molecules have been engineered into diverse peptide arrays for presentation to T cells as part of the mechanism for recognition of autologous and non-autologous cells and pathologically altered cells. Join. A detailed analysis of peptides bound to soluble HLA-G and membrane HLA-G proteins shows that, like MHC class 1, HLA-G also binds to diverse but less complex peptide arrays (Lee et al. , 1995). Some of these peptides are derived from intracellular proteins that, along with MHC molecules, are found in blood mononuclear cells (eg, T cells).
Cell) constitutes a minor histocompatibility antigen that elicits an immune response. HL
A-binding peptides can be easily fractionated, fully or partially purified, and sequenced, and by measuring their ability to reconstitute lysis of target cells by cytotoxic T cells It can be assayed for its ability to elicit a response (den Haan et al., 1998).

The complete gene sequence of HLA-G is known, and DNA sequence analysis of HLA-G has shown that the HLA-G gene exhibits restricted polymorphisms. van
der Van and Ober, 1995 tested the first 6 exons of HLA-G in 45 healthy African Americans and observed changes in exons 2 and 3. This corresponds to the α1 and α2 domains of the peptide binding groove. The most common polymorphism observed is the C to T transition at position 1488, which corresponds to codon 93. Another common polymorphism is Harrison et al., 1
Identified by 993, which is 14 bp in exon 8 of the gene
It is a deletion. These results indicate that HLA-G is a polymorphic gene that can potentially display a wide variety of peptides. The pattern of variability in HLA-G resembles that of other class I MHC genes, in which amino acid substitutions are clustered in the α1 and α2 domains.

Three observations of altered expression of HLA-G in preeclampsia have been reported.
Colbern et al., 1994, HLA-G levels in placental tissues are reduced in pre-eclampsia, and reduced expression appears to be associated with a decreased number of trophoblasts in pre-eclamptic placental tissues. showed that. Hara et al., 1996 showed that in preeclamptic patients, the extraciliary trophoblast clusters were depleted of HLA-G. Examination of human pre-transplant blasts showed that only 40% of the blasts express HLA-G (Jurisicova et al. 1996).

Hereditary Body of evidence shows that preeclampsia and eclampsia are largely under genetic control. However, the underlying genetic mechanism of susceptibility to preeclampsia remains unclear. This is due, in large part, to the disruptive factors characteristic of its heritability. First, this condition is specific to pregnancy, and previous genetic studies have shown that the causative gene acts through the maternal or fetal genotype, or through some interaction between the two. It was not possible to clarify whether or not to do it. Second, preeclampsia is large and is constrained to primagravidas, which has a much lower onset in subsequent pregnancies, and third, because the condition is pregnancy-specific, the genetic distribution in men is Difficult to evaluate.

The diagnosis of true preeclampsia can be complicated by other hypertensive disorders, such as intrinsic hypertension and hypertension resulting from renal disease. Such hypertensive disorders, unlike true preeclampsia, nevertheless can confuse the diagnosis and thus pose a problem for genetic studies.

The classification of preeclampsia by several investigators as a disease of immune dysfunction has prompted numerous studies on the role of the major histocompatibility complex in the genetics of preeclampsia.

[0016] There are numerous published studies on HLA association with preeclampsia (Coop.
er et al., 1993). Besides the fact that this positive association, with the exclusion of one, has not been reproduced in studies by others, these studies are subject to other difficulties. The number of individuals is generally small compared to multiple antigens for each of the HLA loci. Only significant associations tend to be reported, and in addition to the associations reported here, there may be complicated studies that do not show unpublished associations. The first four associations reported are associated with antigen sharing and homozygosity. The number of recognized antigens increased significantly over time. Antigens are split as new sera become available, and the use of DNA technology has shown that more than 100 HLA-A and 100 HLA-B alleles and more than 25 HLA-DRB alleles (serum as DR4 These were subdivided so that there were 5 distinct common sequences that were recognized biologically). Therefore, those typed as homozygous in homozygous or shared antigens in earlier studies cannot be relied upon to be homologous in sequence or function. Detecting homozygotes with sera in early studies suffers from the particular difficulty of distinguishing them from heterozygotes for another allele where serum is absent (blank).

At least three studies have further investigated the association between preeclampsia and HLA-DR by linkage analysis (Winton et al., 1990; Hayward et al., 199).
2; Harrison et al., 1997). In these final studies, no evidence was found for linkage of the HLA region to preeclampsia. Hayward
(1992) also investigated several candidate genes and random DNA markers. Overall, no evidence was found for linkage to some candidate genes shown for hypertensive pathogenesis, and their results ruled out linkage to some markers. In these studies an autosomal recessive model was postulated. Winton et al. (1990) also analyzed their data for HLA linkage using the affected kin pair method and the affected lineage method. Neither of these methods made any assumptions about the mode of inheritance and neither showed signs of linkage. Most cases of preeclampsia are considered idiopathic. Familial pregnancy-induced hypertension has been described, and two loci have been shown in the familial form of the disorder (ie, candidate region on chromosome 4 and eN on chromosome 7).
OS gene region) (Harrison et al., 1997, Arngrimsson et al., 1997). The epidemiology of PET is consistent with familial pregnancy-induced hypertension and idiopathic PET is different.

[0018] Humphrey et al., 1995 investigated HLA-G deletion polymorphisms for association with preeclampsia. Specifically, preeclamptic patients, offspring of preeclamptic mothers, relatives of preeclamptic patients, preeclamptic husbands, and normal control groups were genotyped for other types. Preeclampsia and HLA- in the offspring of the mother or preeclampsia mother
There was no detectable association with the G deletion polymorphism.

[0019] Karhukorpi et al., 1997, H for its association with recurrent sudden pregnancy.
The LA-G polymorphism was investigated. Specifically, they showed that there was no association between some HLA-G restriction fragment length polymorphisms and recurrent spontaneous pregnancy.

In the largest study of monozygotic twins, preeclampsia was reported in the first pregnancies of 5 and all affected mothers were inconsistent with their twins. A second well-documented report of the same twin set also showed a clear discord in their preeclampsia in their first pregnancy. These findings challenge the recessive model and further support a role for fetal paternal genotype in disorders. Moreover, although subject to some debate, preeclampsia occurs in mothers with monozygotic and dizygotic twins, advocates for recessive fetal genotypes, and favors dominant paternal genes in the fetus. And discuss.

[0021] Several studies have considered the possibility of altering paternity as a contributing factor in the development of preeclampsia in people who have had multiple births. Most notably, a strong association between preeclampsia and paternal changes was observed (Lie et al., 1998).

Much of the research on preeclampsia is based on the hypothesis of a major susceptibility locus in affected mothers, and almost all genetic studies to date have demonstrated that between maternal genotype and preeclampsia. Focus on chains or associations. Fetal HLA-G
To test the hypothesis that it is the most likely candidate gene for the disorder, we investigated the HLA-G genotype in the preeclamptic and control triads and found that HLA-G is a normal pregnancy. It was shown to be linked to both outcome and preeclamptic pregnancy outcomes and was associated with recurrent spontaneous abortion. We also found that HLA- in the second trimester of the control triad and the preeclamptic triad.
G genotype was investigated and specific HLA-G in the fetus at first pregnancy
The presence of the allele was shown to allow the development of different HLA-G alleles in the second pregnancy. This means that HLA-G may induce tolerance to the antigen in the first pregnancy and / or is selected and / or causes pregnancy-related disorders in the first pregnancy. Not 2
We show that the maternal immune system can be modified to accept the fetus at the second pregnancy.

Pregnancy loss is the most common complication of human gestation in pregnant women. Most of these losses are clinically unrecognized.
Using a very sensitive assay, the total incidence of miscarriage was estimated to be 31%, which occurred in very early pregnancies (ie, before the pregnancy was clinically recognized). Includes a 22% loss (Wilcox et al., 1988). Recurrent spontaneous abortion (RSA) or recurrent abortion (defined as a loss of third or more sudden pregnancies before 20 weeks gestation) occurs in less than 1% of pregnant women. Studies suggest that the chance of successful pregnancy in naive untreated women who have had more than one first trimester is approximately 30% -50%. It is generally accepted that RSA is a condition with many different causes, but about 50% of all RSA cases are not explained by structural, endocrine, infectious, or anatomical factors. Awareness that recurrent pregnancy losses can have autoimmune causes (immunity against self) and alloimmunity causes (immunity against others) even in women without clinically diagnosed autoimmune diseases within the last few years. Is increasing. This is H
Much emphasis has been placed on investigating the role of the LA system and RSA, especially on the extent of HLA allele and haplotype sharing between RSA couples. Fetuses in which the HLA allele does not differ from the maternal allele (ie, histocompatibility fetus) are H
Fetuses in which the LA allele differs from the maternal allele (ie, a tissue-incompatible fetus)
It has been suggested that they are more likely to have miscarriages. A couple who is matched for the HLA allele or haplotype will then develop a histocompatibility fetus and thus be at risk of miscarriage.

[0024] Ober et al. (1998) conducted a 10-year expected study of HLA matching and pregnancy outcomes. A significant increase in fetal loss was observed in couples who were matched for 16 locus haplotypes that included the entire HLA locus. Ch
ristiansen et al. (1997) tested HLA-C and HLA-Bw in an unexplained RSA couple. They found no change in HLA-C, but a significantly higher number of R than control couples.
SA couple has the HLA-Bw4 haplotype. Jin et al. (1995) examined the degree of sharing of HLA-A, HLA-B, HLA-DR and HLA-DQ haplotypes. They found a significant excess of HLA-DR sharing in married couples with RSA and a significant excess of HLA-DQ sharing in unexplained infertile couples.

Several groups recorded conflicting results. Billingard et al.
(1995) examined allelic sharing of HLA-A, HLA-B, and HLA-DR, and found no higher degree of HLA sharing in couples with RSA than in infertile couples. . Caudle et al., (
1983) reported similar findings. HLA-A, HLA-B, and HLA
-DR alleles were typed in a large population of unexplained RSA couples and in control couples (Sbracia et al., 1996).
. No increased sharing of HLA alleles was seen. Furthermore, there was no difference in the frequency of HLA alleles between the RSA and control couples. Saski et al. (1997) reported an increased frequency of the HLA-DR4 allele in women with RSA compared to control women.

The role of HLA sharing as a risk factor for RSA remains controversial, and no studies have reported diagnostic or prognostic significance for HLA sharing in individual couples. Moreover, reports of significant sharing of class II genes are difficult to explain. This is because fetal cells that come into contact with the maternal immune system during pregnancy lack HLA class II expression.

According to the invention, a method for diagnosing normal pregnancy, susceptibility to preeclampsia and / or eclampsia and / or intrauterine growth retardation, and / or susceptibility to miscarriage and / or miscarriage-related infertility. Provided. The method comprises the steps of: a) obtaining a fluid and / or tissue sample from a female and / or male and / or fetus; and any of the following b) HLA-G nucleic acid and / or HLA-G. Determining the sequence of all or part of the linked nucleic acid; or c) detecting variant forms of all or part of the HLA-G protein and / or the protein encoded by the HLA-G linked gene; or d. ) Measuring the functional activity of all or part of the HLA-G encoded protein and / or the protein encoded by the HLA-G linked gene; or e) transcribed from the HLA-G mRNA or HLA-G linked gene. MRN
Measuring the size and / or level of all or part of A; or f) measuring the size and / or level of all or part of the HLA-G protein or the protein encoded by the HLA-G linked gene. Or g) quantifying cells or molecules whose concentrations are altered as a result of HLA-G action; and h) any of parameters b) to g) for normal pregnancy and / or preeclampsia and Comparing with female and / or male and / or fetal parameters of pregnancy with / or eclampsia and / or intrauterine growth retardation and / or susceptibility to miscarriage and / or miscarriage-related infertility results.

Preferably, the HLA-G nucleic acid is C and / or codon 93 in exon 3.
Alternatively, it is analyzed for the presence of T alleles and / or exon 8 insertion and / or deletion alleles.

Preferably, the functional activity of HLA-G and / or HLA-G mRNA
And / or the effect of one or more HLA-G sequence variants on the size and / or level of all or part of the encoded peptide is measured.

Most simply, the present invention provides a method of diagnosing susceptibility to pre-eclampsia and / or eclampsia and / or intrauterine growth retardation and / or susceptibility to miscarriage and / or miscarriage-related infertility. The method comprises the steps of: a) obtaining nucleic acid from a patient and / or a promising parent and / or fetus; b) analyzing the nucleic acid isolated in step a), Or establishing a HLA-G sequence variant present in the fetus; and c) comparing the known HLA-G sequence variant with the HLA-G sequence variant identified in step b).

Preferably, the HLA-G sequence variant is a DNA sequencing, PCR restriction fragment length polymorphism analysis, glycosylase-mediated polymorphism detection, oligonucleotide hybridization, gel electrophoresis detection of polymorphisms, amplification-based detection approach. It is established by characterizing all or part of the DNA sequence of the HLA-G gene by a method selected from

Suitably, a layered approach is used whereby the C / T-93 in exon 3 and the insertion / deletion polymorphism in exon 8 are first genotyped, followed by exon 3, Other changes in exon 2, intron 2 are followed by exons 1 and 4, followed by the remaining genotyping of the HLA-G gene.

The invention also provides a kit for diagnosing normal pregnancy, preeclampsia and / or susceptibility to eclampsia and / or intrauterine growth retardation and / or susceptibility to miscarriage and / or miscarriage-related infertility. . The kit comprises: a) an oligonucleotide primer for the amplification of all or part of the HLA-G gene and / or HLA-G linked DNA; b) a DNA / RNA polymerase, reverse transcriptase, deoxyribonucleotide d
Amplification reagents for amplification of genomic DNA and / or RNA segments selected from ATP, dCTP, dGTP, dTTP and dUTP, and / or ribonucleotide ATP, CTP, GTP, TTP and UTP, and reaction buffers; c ) Reagents for identifying sequence variants in DNA and / or RNA; d) Control DNA and / or RNA.

Preferably, the primer of (a) is HLA-using the polymerase chain reaction.
Allows specific amplification of all or part of the G gene. Some polymorphisms are H
It is known to occur in the LA-G gene. The C to T polymorphism occurs at nucleotide 1488 at the third position of codon 93, which is referred to herein as C / T-93. C-93 is one allele of the polymorphism, T
-93 is another allele of the polymorphism. Suitably, the C / T-93 polymorphism uses the primer 5'-TACTCCCGAGTCTCCGGGTCTG-3 '(SEQ ID NO: 1) as the forward primer and 5'-AGGGCGCCCCACTGCC.
Amplified C-93 allele (SEQ ID NO: 4) and amplified T-93 allele (SEQ ID NO: 4) by PCR amplification of a fragment of intron 2-exon 3 using CCTGGTAC-3 ′ (SEQ ID NO: 2) as a reverse primer. No. 5), followed by the forward primer 5′-GACCGAGGGGGTGGGGCCAGGGTT.
CT-3 '(SEQ ID NO: 3) and reverse primer 5'-AGGGCGCCCCA
Genotyped by semi-nested PCR amplification with CTGCCCCTGGTAC-3 '(SEQ ID NO: 1). In the half-nested amplification reaction, dTTP is d
Replaced by UTP. The 3'end of the forward primer is designed such that the first U incorporated downstream of the forward primer is at or at the polymorphic site of codon 93. Amplification with an end-labeled forward primer is followed by glycosylase-mediated cleavage of the amplification product. The cleavage product was subjected to denaturing gel electrophoresis (2
0% polyacrylamide) and visualized by autoradiography. The C-93 allele is a 32n fragment (SEQ ID NO: 8).
), The T-93 allele is detected as a 27n fragment (SEQ ID NO: 9).
Detected as.

The common 14 base pair insertion / deletion polymorphism in exon 8 of the HLA-G gene is referred to herein as I / D-E8, where I-E8 refers to that polymorphism. Is one allele of the polymorphism, and D-E8 is another allele of the polymorphism. Suitably, genotyping of the HLA-G exon 8 deletion polymorphism is performed by amplifying a short section flanking the exon 8 deletion site. This is accomplished by using the polymerase chain reaction with primers designed to hybridize to the known DNA sequence of exon 8. The forward primer was 5'-TGTGAAACAGCTGCCCGTGT-3 '(SEQ ID NO: 10).
), And the reverse primer is 5'-AAGGAATGCAGTTCAGCA.
It is TGA-3 '(SEQ ID NO: 11). The I / D exon 8 polymorphism is genotyped by size separation of PCR products on a 10% non-denaturing polyacrylamide gel and visualized by staining with ethidium bromide. The I-E8 insertion allele yields a 151 bp product (SEQ ID NO: 12) and the D-E8 deletion allele yields a 137 bp product (SEQ ID NO: 13).

Suitably, allele-specific genotyping includes maternal and paternal C / T-9
3 as well as HLA-G of ID-E8 are performed when haplotypes cannot be directly assigned. This is achieved by using allele-specific primers that allow selective amplification of the I-E8 or D-E8 alleles. Following specific amplification of the allele, the C / T-93 polymorphism was then identified by the above-mentioned GMPD.
Genotyped using the assay. Primers for amplification of the I-E8 allele were 5'-TACTCCCGAGTCTCCGGGTCTG-3 '(SEQ ID NO: 1) (forward primer) and 5'-CAAAGGGAAGGCATGA.
ACAAATCTTG-3 '(SEQ ID NO: 14) (reverse primer). D
The primer for amplification of the -E8 allele is 5'-TACTCCCGAGT
CTCCGGGTCTG-3 ′ (SEQ ID NO: 1) (forward primer) and 5 ′
-GTTCTTGAAGTCACAAAGGGACTTG-3 '(SEQ ID NO: 15
) (Reverse primer). Four such allele-specific primers are
Two possible haplotypes: I-E8 and C-93 haplotypes (SEQ ID NO: 16), I-E8 and T-93 haplotypes (SEQ ID NO: 17), D-E
8 and C-93 haplotype (SEQ ID NO: 18), and D-E8 and T-
Yields 93 haplotypes (SEQ ID NO: 19).

Suitably the haplotypes are constructed and, suitably, alleles transmitted and not transmitted to the progeny / fetus are assigned.

[0038] Preferably, the amplification reagent is a thermostable DNA polymerase, an amplification buffer, and D.
The NA precursor nucleotide is shown.

All and part of any HLA-G and / or HLA-G linked sequences are also nucleic acid sequence-based amplification, self-maintained sequence replication, transcription-mediated amplification, strand displacement amplification, and ligase. It can be amplified by a method selected from a chain reaction, or a combination of those methods.

Preferably, the comparison of the identified one or more variants is performed by association and / or linkage analysis and / or transmission analysis. Preferably all or part of the HLA-G sequence is cloned into the vector.

The invention may include: a) obtaining a nucleic acid or body fluid or tissue sample from a parent and / or a planned parent and / or fetus; b) isolated in step (a). Establishing the HLA genotype or serotype of the parent / destined parent and / or fetus by analyzing the nucleic acid or body fluid or tissue sample; c) the HLA genotype or serum identified in step (b). The molds are known H
Comparing to the LA genotype or serotype.

Preferably, the method comprises the step of measuring cellular and / or soluble HLA-G levels. Preferably, HLA-G levels are measured by immunoassay using an antibody against the particular HLA-G protein.

The present invention may include the step of identifying a variant form of the HLA-G protein and / or its level of presence in a sample. Preferably, the HLA-G variant protein and / or its level is detected and / or quantified by an immunoassay using specific antibodies that detect the HLA-G variant or HLA-G protein. Alternatively, antibodies specific for HLA-G protein variants and / or electrophoretic and / or chromatographic separation methods can be used. Preferred methods for detecting HLA-G proteins and variants thereof are enzyme-linked immunoassay (ELISA), radioimmunoassay (RIA), immunoradiometric assay (IRMA), and immunoenzymatic assay (IEM).
These include sandwich assays using monoclonal and / or polyclonal antibodies, including A).

The present invention may include the step of measuring the level of a molecule whose concentration changes as a direct and / or indirect result of HLA-G action. Preferably, these molecules are selected from IL-1, IL-2, IL-3, IL-4, IL-6, IL-10β, and tumor necrosis factor α. Preferably, the level of such molecules is
It is measured by immunoassay using antibodies specific for those molecules.

Alternatively, the method may include measuring the level of a trophoblast specific marker. Preferably, the trophoblast markers are cytokeratin pregnancy-specific glycoprotein 1, human chorionic gonadotropin, and human placental lactogen. Preferably, the levels of such molecules are measured by immunoassay using antibodies specific for these molecules.

In one embodiment, the method may include the following steps: a) blood mononuclear cells and / or a subset of such cells in one or more HL.
AG variants and / or any combination thereof, and / or HL
Incubating with a cell expressing all or part of one or more variants of the AG gene and / or a combination of one or more variants thereof, wherein blood mononuclear cells and / or HLA -The G variant is of female and / or male and / or fetal origin. b) blood mononuclear cells, and / or HLA-G, and / or one or more HLA
-A step of analyzing the activity of cells expressing the G variant.

Preferably, the blood mononuclear cells are obtained as a blood sample and / or a tissue sample from a female and / or the female blood mononuclear cells are derived from a donor and / or cell line panel. Obtained through matching cells. Preferably, a population of T cells and / or NK cells is isolated from a blood sample by density centrifugation and / or immunoselection. Preferably, the blood mononuclear cells that match the female blood mononuclear cells have the female HLA serotypes and / or expanded HLA genotypes and / or HLA-G genotypes as HLA of blood mononuclear cells. Serotype and / or expanded HLA genotype and / or HLA
-Identified from the test panel by matching the G genotype. Preferably, the HLA-G that matches the male and / or female HLA-G is the male and / or female.
Or a female HLA-G type and / or HLA-G genotype with an HLA-G type and / or HLA-G genotype of an HLA-G protein and / or one or more HLA-G gene modifications in a test panel thereof. Identified from the test panel by matching with cells expressing the body. Preferably, such a test panel is assembled by growing cells expressing one or more HLA-G variants. Such cells may be derived from natural tissues such as placenta and / or HL
Manufactured artificially by introducing one or more vectors having HLA-G gene variants capable of promoting expression of the A-G gene into cells and / or inducing expression of native HLA-G in cells. . Suitably the vector used is a plasmid,
Based on phage, virus, and / or artificial chromosomes. Preferably HLA
-G protein is used as a crude preparation and / or is wholly or partially purified from such cells. The HLA-G protein can be loaded with naturally or artificially bound peptides.

Preferably, the HLA-G-blood mononuclear cell interaction is measured by assessing activation of blood mononuclear cells, which comprises one or more of the following; cell proliferation, transformed cytotoxicity. Response, surface marker expression, cytokine production, conjugate formation and target specificity.

The method may include the steps of: a) cloning the HLA-G gene from the parent and / or the expected parent and / or fetus; b) the gene cloned in vitro and / or in vivo. To HLA-G
Expressing the protein; c) measuring the level of activity of the expressed HLA-G protein; d) changing the level of activity of the expressed HLA-G to that of the activity observed against normal HLA-G protein. The process of comparing with the level. The method also includes: a) H present in a sample from a female and / or a male and / or a fetus.
Establishing all or part of the LA-G sequence and / or the HLA-G linked sequence by analyzing the nucleic acid from the sample; b) any one or more variants identified in step (a). Or a comparative analysis of whether or not any combination thereof shows susceptibility to normal pregnancy or preeclampsia and / or eclampsia and / or intrauterine growth retardation, and / or abortion and / or abortion-related infertility. And / or one or more variants of HLA-
Determining by analyzing the effect on functional activity of G and / or the effect on HLA-G mRNA.

Preferably, the HLA-G sequence variant is the HLA-G gene and / or contiguous linked DNA (HLA-A, HLA-B, HLA-C, HLA-E, HLA-).
(Including the F and HLA-H genes).
-G or established by amplifying all or part of the contiguous linked DNA and characterizing by identifying the sequence variants present using one or more sequence modification detection methods.

Suitably, one or more copies of all or part of the HLA-G gene may be transferred to several amplification approaches (eg polymerase chain reaction (PCR), nucleic acid sequence based amplification (NASBA), self Amplification by either sustained sequence replication (3SR), transcription-mediated amplification (TMA), and strand displacement amplification.Amplification of target nucleic acid molecules also includes ligase chain reaction (LCR), short PCR step (PLCR). Can be carried out by using a modified method of LCR using, suitably DNA or mRNA is used as amplification substrate, suitably mRNA is converted to DNA using reverse transcriptase. Suitably, the amplified molecule can be directly analyzed and / or cloned into a vector to facilitate analysis.
Suitably the DNA sequence modifications are detected by a variety of genetic modification detection methods, including any one or more of the following; DNA sequencing, glycosylase mediated polymorphism detection, restriction fragment length polymorphism analysis, enzymatic or Chemical cleavage assays, hybridization to DNA probe arrays, allele-specific oligonucleotide hybridization assays, allele-specific amplification methods (eg, amplification refractory method (ARMS), migration-based polymorphic electricity based on gel matrix) Electrophoretic detection,
5'nuclease assay, and ligase chain reaction.

Suitably, the one or more variants identified are sensitive to normal pregnancy or to preeclampsia and / or eclampsia and / or intrauterine growth retardation, and / or susceptibility to miscarriage and / or miscarriage-related infertility. It can be determined whether or not there are known variants of interest. Alternatively, the HLA-G variant and / or HLA-g linked variant is susceptible to normal pregnancy or preeclampsia and / or eclampsia and / or intrauterine growth retardation, and / or susceptibility to miscarriage and / or miscarriage-related infertility. Competitive analysis is performed by gene association and / or gene linkage methods to determine whether or not to be associated with.

Alternatively, the HLA-G variants associated with susceptibility to normal pregnancy and / or preeclampsia and / or eclampsia and / or intrauterine growth retardation, and / or abortion and / or miscarriage-related infertility are HLA-GLA. -HL of G variant
It can be identified by its effect on AG function. Suitably, the HLA-G sequence variant is a measure of the interaction of one or more HLA-G variants and / or any combination thereof with blood mononuclear cells, and / or of HLA-G messenger RNA. Size and level, and / or size and level of HLA-G gene product, and / or peptide binding to one or more HLA-G variants,
And / or can be functionally analyzed by measuring any combination thereof. HLA-G-blood mononuclear cell activity is measured by assessing activation of blood mononuclear cells including one or more of the following assessments; cell proliferation, cytotoxic response, surface marker expression, cytokine production, Conjugate formation and target specificity.

The present invention also relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the following, optionally together with a pharmaceutically acceptable carrier or excipient; HLA-G protein, and / or Or HLA-G expressing cells, and / or one or more peptides that bind to HLA-G, and / or donor-derived blood mononuclear cells, and / or known to interact with HLA-G variants. Cells from the test panel,
Cytokines and any combination thereof (IL-1β, IL-2, IL-
3, IL-4, IL-6, IL-10, and tumor necrosis factor α, and / or cytokine inhibitors and / or tumor necrosis factor α inhibitors, and / or cytokine derivatives and / or tumor necrosis factor α Including derivatives).

The present invention also provides methods of screening for agents that could potentially be used as diagnostic indicators and / or drug targets for pre-eclampsia, miscarriage, miscarriage-related infertility, and intrauterine growth retardation. a) HLA- after interaction with HLA-G and / or HLA-G expressing cells
Step b) measuring the expression level of one or more genes and / or proteins of G-expressing cells and / or blood mononuclear cells and / or T cells and / or a subset of their natural killer cells, identified in step (a) Expression level after interaction with HLA-G and HLA-G expressing cells in normal and / or preeclamptic and / or intrauterine growth retarded and / or miscarriage and / or miscarriage related infertility Of HLA-G expressing cells and / or blood mononuclear cells and / or T cells and / or their natural killer cell subsets.

In a further aspect, the invention provides a method of screening for a therapeutic agent for preeclampsia, and eclampsia, and intrauterine growth retardation and miscarriage, and miscarriage-related infertility, the method being selected from: A) identifying agents that alter HLA-G expression; b) identifying agents that alter HLA-G activity; c) identifying agents that mimic the effects of HLA-G; d) identifying agents that bind to HLA-G; e) identifying peptides that bind to HLA-G; f) identifying agents that bind to HLA-G receptors; g) interacting with HLA-G. Cell mass in acting HLA-G expressing cells and / or blood mononuclear cells and / or in HLA-G expressing cells interacting with blood mononuclear cells. Identifying cells expressed using a DNA probe array in the background; h) the expression interacts with and / or in response to HLA-G expression in the cells. Identifying genes expressed using a DNA probe array in the background of the cells that are altered in response to i) is expressed in HLA-G expressing cells and / or blood mononuclear cells that interact with HLA-G and / or in HLA-G expressing cells that interact with blood mononuclear cells using mass spectrometry. Identifying the identified protein.

Preferably, sperm and / or semen and / or female reproductive tissue are screened for agents such as: a) agents that alter HLA-G expression in fertilized eggs and / or embryos; b) An agent that alters the cell division rate of fertilized eggs and / or embryos; c) induces a cellular factor in cells in culture and / or cells in vivo that alters the cell division rate of fertilized eggs and / or embryos Drug.

The method may include: a) HLA-G expressing cells and / or blood mononuclear cells and / or T cells and / or them after interaction with HLA-G and HLA-G expressing cells. Measuring the expression level of one or more genes and / or proteins of a subset of natural killer cells of b), the expression level identified in step (a) being compared with normal pregnancy and / or preeclamptic pregnancy and / or in utero HLA-G expressing cells and / or blood mononuclear cells and / or after interaction with HLA-G and HLA-G expressing cells associated with growth retarded pregnancy and / or miscarriage pregnancy and / or miscarriage related infertility Comparing with the expression level of a subset of T cells and / or their natural killer cells.

Preferably, blood mononuclear cells and / or HLA-G expressing cells are tested for female and / or male and / or foetus, and / or blood mononuclear cells and / or HLA-G expressing cells. Obtained from the panel.

Preferably, gene expression is measured by any one of a number of methods or a combination thereof, including: cDNA and / or cDNA derived from those cells.
Or RNA and hybridization between DNA probe and / or RNA probe and / or DNA probe array, quantitative (reverse transcriptase polymerase chain reaction) RT-PCR, 5 ′ nuclease assay, ribonuclease protection assay and S1 nuclease assay Quantitative amplification approach such as.

Preferably, protein expression is measured by any one or a combination of several methods including: one-dimensional and / or two-dimensional gel electrophoresis and protein staining, and / or protein. Enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay (IRMA), and immunoenzymatic assay (IEMA), which are sandwich assays and Western blotting using monoclonal and / or polyclonal antibodies Detection of one or more proteins using

Alternatively, the method may include: a) measuring the expression level of one or more genes and / or proteins in cells expressing HLA-G; and b) identified in step (a). Comparing the expression level to the expression level in cells that do not express HLA-G.

[0063]   Preferably, the cell is a fertilized egg of an animal and / or an embryo of an animal.

The present 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: Provided: a) A pharmaceutically effective amount of an effective HLA-G protein and / or HLA-G
Treating a woman with all or part of cells expressing a peptide that binds to and / or HLA-G; b) its level or activity is directly or indirectly altered by HLA-G, Treating females with all or part of a pharmaceutically effective amount of a molecule or inhibitor of molecules; c) treating females with all or part of a pharmaceutically effective amount of an agent that alters HLA-G expression. D) treating a woman with all or part of a pharmaceutically effective amount of an agent that alters NK cell activity; e) a pharmaceutically effective amount of an agent that mimics all or part of HLA-G action, Treating women with all or part; f) treating women with all or part of a pharmaceutically effective amount of a molecule that inhibits the interaction between HLA-G and one or more of its receptors. ; G) H Treating a woman with all or part of a pharmaceutically effective amount of an agent that alters the size and / or level of AG mRNA; h) alters the activity of blood mononuclear cells associated with HLA-G. Treating a woman with all or part of a pharmaceutically effective amount of a drug; i) treating the woman with blood mononuclear cells that recognize foetal and / or autologous HLA-G; j) treating a woman with cells expressing HLA-G protein and / or HLA-G.

The invention may include: a) a subset of blood mononuclear cells and / or their T cells and / or natural killer cells from a female and / or male and / or fetal and / or test panel and And / or obtaining HLA-G and / or HLA-G expressing cells; b) HLA- after interaction with HLA-G and / or HLA-G expressing cells.
Measuring the expression levels of one or more genes and / or proteins in G-expressing cells and / or blood mononuclear cells; c) comparing the expression levels identified in step (b) to normal and / or preeclamptic pregnancy and And / or comparing its expression level in blood mononuclear cells and / or HLA-G expressing cells in intrauterine growth retarded pregnancy and / or miscarriage pregnancy and / or miscarriage related infertility.

Preferably, the blood mononuclear cells and / or HLA-G expressing cells are obtained as a blood sample and / or a tissue sample. Preferably, a population of T cells and / or NK cells is isolated from the blood sample by density centrifugation and / or immunoselection. Preferably, HLA-G expressing cells are isolated by immunoselection.

The invention also provides a method for improving the outcome of fertilization and pregnancy, wherein a male and / or female partner and / or sperm and / or egg and / or fertilized egg recipient and And / or zygotes and / or embryos whose genotype and / or serotype are associated with normal pregnancy outcomes and / or pre-eclampsia and / or eclampsia and / or intrauterine growth retardation, And / or is selected based on HLA-G such that it is not associated with susceptibility to miscarriage and / or miscarriage-related infertility.

In particular, a method for improving the success of pregnancy selected from the following is provided: a) Mating with men of different HLA-G genotypes, and / or different HLA-.
Prior to in vitro fertilization using male sperm of the G genotype and / or embryo transfer where the male HLA-G is a different HLA-G genotype, the known HLA
-Pre-treating a woman with sperm and / or its attenuated form, and / or semen and / or a fraction thereof, from a male with a G genotype. b) Prior to in vitro fertilization, spermatozoa of known HLA-G genotype were treated with different H
Mixing with sperm and / or its attenuated form, and / or semen and / or fractions thereof, from a male having the LA-G genotype.

Preferably, the outcome of fertilization and / or pregnancy is (a) their HLA-G and / or HLA genotype and / or serotype, or (b) their HLA-G and / or HLA-G. The activity of blood mononuclear cells that interacts with normal pregnancy results and / or preeclampsia and / or eclampsia and / or intrauterine growth retardation and / or miscarriage and / or miscarriage-related Improved by selection of male and / or female partner and / or sperm and / or sperm and / or egg and / or fertilized egg recipient and / or zygote and / or embryo so as not to be associated with susceptibility to infertility It

Cloning of all or part of one or more HLA-G genes in any of the above methods can amplify all or part of one or more HLA-G genes and express the inserted gene. This can be achieved by inserting all or part of the amplification product into the vector. Expression of the HLA-G protein from the gene cloned in any of the above methods can be achieved by introducing the expression vector into a suitable host, such as a eukaryotic cell in bacteria or culture. The level of activity of the HLA-G protein expressed in any of the above methods is a) HLA-
Measuring directly or indirectly the interaction of cells expressing G protein and / or HLA-G protein with blood mononuclear cells, and / or b) the level of which is HLA-G protein and / or HLA- Detecting one or more molecules that are altered as a result of the interaction of the G protein expressing cells with blood mononuclear cells, and / or c) the HLA-G protein and / or HLA-G protein expressing cells, It can be achieved by measuring changes in cell division rate due to direct and / or indirect effects on blood mononuclear cells.

HLA-G, as defined herein, refers to any form of HLA-G, as well as different isoforms of HLA-G resulting from alternative splicing pathways, combinations of different HLA-G isoforms, and secretion. HLA-G, membrane-bound HL
Refers to any complex comprising HLA-G, including AG, HLA-G having a peptide bond, and HLA-G bound to β-2-microglobulin. HLA-G
Protein refers to any crude, partially purified and / or fully purified form of HLA-G.

The invention also provides for the diagnosis of, or 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. Of a potential diagnostic indicator and / or drug target in a method for screening a potential therapeutic agent for use in the manufacture of a medicament for monitoring the progress of pregnancy in a test kit for For the prevention of preeclampsia and / or eclampsia and / or intrauterine growth retardation and / or abortion and / or miscarriage-related infertility. 1-2 for monitoring the progress of pregnancy in There is provided the use of a DNA sequence selected from any one of 1.

The present invention also provides a method for inducing tolerance to non-self tissue in a host, which method comprises HLA-G, and / or a peptide derived from non-self tissue, and / or non-self tissue. Loaded with HLA-G expressing cells that are derived or associated with non-self tissue and / or non-self tissue with the HLA-G gene introduced so that HLA-G is expressed in all or some tissues HLA
-Comprising the step of administering G.

In a further aspect, the invention provides a method for treating autoimmunity. This method involves HLA-G, and / or HLA loaded with peptides derived from self or non-self tissue and / or specific autoimmune antigens.
-G, and / or HLA derived from self-organization and / or non-self-organization
-G expressing cells, and / or administering HLA-G with autologous tissue and / or non-self tissue carrying the HLA-G gene introduced such that it is expressed in all or part of the tissue.

(Method) (Identification of Subject) At the start of sampling, deterioration (deteriorat) in pregnancy
Ion) is an index of preeclampsia, so the patient with preeclampsia was identified as a primigravida who delivered by Caesarean section at or before the 36th week of pregnancy. Diagnostic symptoms include blood pressure increase in the diastolic phase of more than 15 mm Hg or 30 mm from the measurement in early pregnancy
Increased systolic blood pressure over Hg or 140/90 mm in late pregnancy
Elevated blood pressure above Hg, and one or more of the following: proteinuria,
Edema, headache, visual impairment, epigastric pain. Control patients were identified as primiparas with normal childbirth and normal blood pressure. 5-10 ml of blood were obtained from the offspring of preeclamptic first trimesters with informed consent and normal pregnant women.

In the second stage of sampling, a blood sample for DNA extraction and / or
Or cheek swab samples were collected from postnatal tripartite controls. Appropriate informed consent was obtained from all subjects. Control maternals were identified as primiparas <33 years of age with normal childbirth and normal blood pressure. All subjects were of Irish and Caucasian origin. I interviewed my mother and confirmed that she was a primigravida. Identified and sampled triads of preeclamptic primigravidas (first pregnancy) (where mothers have severe preeclampsia) and matched controls of normal primonatal women . Family (mother,
(Father, first and second child) (if this mother has had two or more successful normal pregnancies without pregnancy-related disorders including preeclampsia and miscarriage), and sampling did. Family (mother, father, first and second child) (this mother experiences preeclampsia in her first pregnancy and a normal second pregnancy without preeclampsia and pregnancy-related disorders including miscarriage) Were also identified and sampled. Couples with recurrent spontaneous abortions were also identified and sampled. To minimize the potential for misdiagnosis of PE, we applied strict criteria to sample validation. By definition, spoilage in pregnancy is an indicator of preeclampsia, so a case of preeclampsia was identified as the first pregnancy under 35 years of age delivered by cesarean section at or before 36 weeks gestation. . The diagnostic symptoms are a blood pressure increase in a diastolic phase of more than 15 mm Hg or a systolic blood pressure of more than 30 mm Hg, or a blood pressure increase of more than 140/90 mm Hg in the second trimester, as well as the following 1 More than one: proteinuria, edema,
Headache, visual impairment, upper stomach pain. The diagnostic symptoms were completely normal within 3 months after delivery. A preliminary study of female preeclampsia sisters in this study did not show an increased incidence of the condition. This indicates that preeclampsia in the maternal cohort investigated here is sporadic. We identified a cohort of couples whose mothers had more than two secondary abortions (recurrent abortion).

Genotyping of HLA-G Polymorphisms Genomic DNA was extracted from peripheral blood and / or buccal swab samples by standard methods. When DNA was isolated from blood, the DNA concentration was
The sample was determined by its absorbance at 0 nm. The integrity and purity of this genomic DNA was determined by agarose gel electrophoresis and OD260: OD28, respectively.
It was determined from the ratio of 0.

The C-93T HLA-G polymorphism is also known as the C / T codon 93 polymorphism (and as HLA-G C1488T), and is referred to herein as C / T-93. To be done. Here, C-93 is one allele of the polymorphism and T-93 is the other allele of the polymorphism. To genotype the C / T-93 polymorphism in this genomic DNA sample, exon 3 of the HLA-G gene was cloned using the polymerase chain reaction to a known D flanking exon 3.
It was first amplified using primers designed to hybridize to the NA sequence. The forward primer is 5'-TACTCCCGAGTCTCCGGGTTCG.
-3 '(SEQ ID NO: 1) and reverse primer are 5'-GAGGCGCCCC
ACTGCCCCTGGT-3 '.

Total 25 μl of polymerase chain reaction (100 ng genomic DNA, 50 ng
Each primer of 0.2 mM of each deoxynucleoside triphosphate (dATP, d
CTP, dGTP and dTTP), 50 mM KCl, 10 mM Tris-
HCl, pH 9.0, 25 ° C, 0.1% Triton X-100, 1.5m
M MgCl ₂ , 0.5 U Taq polymerase). The reaction mixture was covered with an equal volume of mineral oil and amplification was carried out on a thermal cycler using the "hot start" technique. Conditions for amplification included denaturation at 94 ° C. for 5 minutes, followed by addition of Taq polymerase. Then 30 cycles were carried out: 94 ° C. for 1 minute, 63 ° C. for 1 minute, 72 ° C. for 1 minute and finally 10 minutes at 72 ° C.
Minute extension.

Genetic determination of the C / T-93 HLA-G polymorphism was then performed using the seminested (s
It was performed using the immuno-nested) amplification approach and the glycosylase-mediated polymorphism detection method (Vauguhan and McCarthy 1998). C /
The 319 bp of the HLA-G gene containing the T-93 polymorphic position was transferred from exon 3 of the pre-amplified HLA-G gene to the exon 3 reverse primer and internal forward using the semi-nested polymerase chain reaction approach. Primer 5'-
Amplification was performed using GACCGAGGGGGTGGGGCCAGGTTTCT-3 '(SEQ ID NO: 3). This forward primer was prepared using polynucleotide kinase with manufacturer's buffer (New England Biolabs) and 5μ.
End labeling was carried out by incubating in Ci 32 P-ATP (3000 Ci / mmol) for 30 minutes at 37 ° C., followed by ethanol precipitation to remove unused labeled nucleotides. Seminested amplification reactions were performed in a total volume of 10 μl (1 μl of 1/500 dilution of pre-amplified exon 3 product, 3 pmoles of forward and reverse primers, 0.2 mM of each deoxynucleoside triphosphate (dATP, dCTP, dGTP). And dUTP), 50 mM KCl, 10 mM Tris-HCl, pH 9.0, 25 ° C., 0.1% Tri.
ton X-100, containing 1.5 mM MgCl ₂ and 0.5 U Taq polymerase). The reaction mixture was covered with an equal volume of mineral oil and amplification was performed on a thermal cycler using the "hot start" technique. Conditions for amplification included denaturation at 94 ° C. for 5 minutes, followed by addition of Taq polymerase. Then 30 cycles were performed: 94 ° C. for 1 minute, 64 ° C. for 1 minute, 72 ° C. for 1 minute.
Min and finally 10 min extension at 72 ° C. The reaction mixture was then treated with exonuclease I to digest the primers that did not extend during the amplification step. This was done by incubating the PCR reaction product with 0.4 units of exonuclease I for 30 minutes at 37 ° C. The exonuclease was subsequently heat inactivated by incubating the reaction for 15 minutes at 80 ° C.

Uracil DNA glycosylase (0.5 units) was then added and the incubation was continued for 1 minute at 37 ° C. After treatment with uracil DNA glycosylase, the AP sites generated in the amplification product were cleaved to completion by adding NaOH to a final concentration of 0.05M and heating the mixture at 95 ° C for 15 minutes. Under these conditions, cleavage occurs on the 5'side of each AP site. The reaction was then neutralized by adding Tris base to a final concentration of 30 mM. Both Exol and UDG, 0.07M Hep
es Dilute with KOH pH 8.0, 1 mM EDTA, 1 mM DTT and 50% glycerol.

An equal amount of formamide added dye (90% formamide, 0.025% bromophenol blue, 0.025% xylene silanol) was added to the sample, which was then heated at 85 ° C. for 5 minutes. The sample was then loaded on a 20% denaturing (7M urea) polyacrylamide gel for size analysis of the cleavage products in this sample and electrophoresis was performed at 60W for 3-4 hours. After electrophoresis, the gel was autoradiographed on an X-ray photographic film at -70 ° C.
It was carried out by exposing for 2 hours. An improved protocol was used during the second stage of genotyping. Essentially, PCR amplification was performed in 25 ml of reaction. Each of these reactions contained 100 ng of genomic DNA, PCR buffer (100 mM Tr
is-HCl, pH 8.3 (20 ° C.), 500 mM KCl, 15 mM MgC
l ₂ ), 200 mM of each dNTP, 300 nM of each primer, and 0.5 U
Taq polymerase from Boehringer. The conditions for amplification of exon 3 were 5'-TACTCCCGAGTCTCC as the forward primer.
GGGTCTG-3 ′ (SEQ ID NO: 1) and 5′-AG as a reverse primer
30 cycles of 94 ° C. for 45 seconds, 61 ° C. for 45 seconds, 72 ° C. for 60 seconds using GCGCCCCACTGCCCCTGGGTAC-3 ′ (SEQ ID NO: 2). This resulted in an amplified C-93 allele (SEQ ID NO: 4) and an amplified T-93 allele (SEQ ID NO: 5). All this sample was then subjected to the recently described glycosylase-mediated polymorphism detection (GMPD) method (Vau
ghan and McCarthy, 1998) using HLA-G C / T
Genotyped for -93 polymorphism. Essentially, the 319 bp fragment was digested with semi-nested PCR to the internal forward primer 5'-GACCGAGG.
It was amplified from exon 3 using GGGTGGGGCCAGGTTTCT-3 '(SEQ ID NO: 3) and the reverse primer 5'-AGGGCGCCCCACTGCCCCTGGTAC-3' (SEQ ID NO: 1). This resulted in an amplified C-93 allele (SEQ ID NO: 6) and an amplified T-93 allele (SEQ ID NO: 7). In the seminested amplification reaction, dTTP was replaced with dUTP. Codon 9
The 3'end of the forward primer was designed so that the first U was incorporated downstream of the forward primer present at or distal to the polymorphic site in 3. Amplification using a P ³² end-labeled forward primer was followed by glycosylase-mediated cleavage of the amplified product. Cleavage products were separated by denaturing gel electrophoresis (20% polyacrylamide gel) and visualized by autoradiography. C-
The 93 allele was detected as a 32n fragment (SEQ ID NO: 8), and T
The -93 allele was detected as a 27n fragment (SEQ ID NO: 9).

A common 14 base pair insertion / deletion polymorphism within exon 8 of the HLA-G gene was identified as
It is referred to herein as I / D-E8 (also known when I-E8 is one allele of the polymorphism and D-E8 is the other allele of the polymorphism). Genotyping of the exon 8 deletion polymorphism in HLA-G was performed by amplifying the short portion of exon 8 adjacent to the deletion site. This was accomplished using the polymerase chain reaction with primers designed to hybridize to known DNA sequences within exon 8. The forward primer is 5'-
TGTGAAACAGCTGCCCGTGT-3 '(SEQ ID NO: 10) and the reverse primer was 5'-AAGGAATGCAGTTCAGCATGA-3'.
(SEQ ID NO: 11) was used.

Polymerase chain reaction was performed in a total volume of 25 μl (100 ng of genomic DNA, 50 n
g of each primer, 0.2 mM of each deoxynucleoside triphosphate (dATP,
dCTP, dGTP and dTTP), 50 mM KCl, 10 mM Tris
-HCl, pH 9.0, 25 ° C, 0.1% Triton X-100, 0.5
in mM MgCl ₂ and 0.5 U Taq polymerase). The reaction mixture was covered with an equal volume of mineral oil and amplification was performed on a thermal cycler using the "hot start" technique. Conditions for amplification included denaturation at 94 ° C. for 5 minutes, followed by addition of Taq polymerase. Then 30 cycles were performed: 9
Extension at 4 ° C for 1 minute, 54 ° C for 1 minute, 72 ° C for 1 minute, and finally 72 ° C for 10 minutes. The I / D exon 8 polymorphism was genotyped by size separation of PCR products on a 10% denaturing polyacrylamide gel and stained with ethidium bromide. The I-E8 insertion allele yielded a 151 bp product (SEQ ID NO: 12) and the D-E8 deletion allele yielded a 137 bp product (SEQ ID NO: 13).

Allele-Specific Genotyping Allele-specific genotyping was performed to gain more information from the transmission of HLA-G polymorphisms in the second-stage study. In the majority of cases, the maternal and paternal C / T-93 and I / D-E8 HLA-G haplotypes could be directly assigned. If all members of the triplicate were heterozygous for either the C / T-93 polymorphism or the I / D-E8 polymorphism, allele-specific amplification was performed to assign haplotypes. . This is the I-E8 allele or D-E8
It was achieved using allele-specific primers that allowed selective amplification of alleles. Following allele-specific amplification, the C / T-93 polymorphism was then genotyped using the GMPD assay described above. The conditions for amplification of the I-E8 allele were 5'-TACTCCCGAG as the forward primer.
Using TCTCCGGGTCTG-3 '(SEQ ID NO: 1) and 5'-CAAAGGGAAGGCCATGAACAAATCTTG-3' (SEQ ID NO: 14) as the reverse primer, 94 ° C for 45 seconds, 64 ° C for 45 seconds, 72 ° C for 6 seconds.
There were 30 cycles of 0 seconds. The conditions for amplification of the D-E8 allele were 5'-TACTCCCGAGTCTCCGGGTCTG- as the forward primer.
3 '(SEQ ID NO: 1), and 5'-GTTCTTGAA as reverse primer
Using GTCACAAAAGGGACTTG-3 ′ (SEQ ID NO: 15), 94 ° C.
For 45 seconds, 56 ° C. for 45 seconds, and 72 ° C. for 60 seconds for 30 cycles. Such allele-specific amplification shows four possible haplotypes: I-E8 and C-93 haplotype (SEQ ID NO: 16), I-E8 and T.
-93 haplotype (SEQ ID NO: 17), D-E8 and C-93 haplotype (
SEQ ID NO: 18) and D-E8 and T-93 haplotypes (SEQ ID NO: 19)
Occurred.

All individuals in both sets of the tripartite were genotyped for some or all of these polymorphisms (Tables 1 and 2) and designated transmitted and non-transmitted alleles, Then he built the haplotype. All individuals
Genotyped for C / T-93 polymorphism and I / D-E8 polymorphism. Comparative statistical analysis was performed. A more detailed analysis was conducted during the second phase of this study. Comparison of allele and haplotype frequencies, and distribution of genotypes for polymorphisms in and between sample cohorts, χ ² contingency table analysis and / or log-linear model analysis and / or transmission. This was done using the imbalance test.

If all members of the triad were heterozygous for the C / T-93 polymorphism and the I / D-E8 polymorphism, allele-specific amplification was performed to determine the haplotype. Therefore, inherited and non-inherited alleles were designated.
This was accomplished using primers that allowed specific amplification of the HLA-G gene from either insertions or deletions in exon 8 to the 5'site of codon 93. The C / T-93 polymorphism was then genotyped at the specifically amplified allele. Using this approach, alleles transmitted and non-transmitted to offspring were designated.

Results All genetic studies to date have examined the maternal genotype of preeclampsia, with the exception of one study on HLA-G deletion polymorphisms. The present inventors have found that fetal HL
The view was that AG is the most likely candidate gene for preeclampsia. A control group of preeclamptic triads (if the offspring were offspring of preeclamptic primigravus) and normal primonatal offspring was studied. 54 preeclamptic offspring and 48 control groups were included in the study.

Genetic analysis of the C / T-93 polymorphism of HLA-G in preeclamptic offspring was
7 cases (13%) homozygous for the C-93 allele, 3 cases (5.6%) homozygous for the T-93 allele and 44 cases (81%) for the heterozygotes.
． 4%). By comparison, control progeny had 18 cases (37.5%) homozygous for the C-93 allele and 8 cases (37.5%) homozygous for the T-93 allele (
16.6%) and 22 cases of heterozygosity (45.8%).

The frequencies of C-93 and T-93 alleles in pre-eclamptic offspring were 0.537 and 0.463, respectively. In comparison, the frequencies in control offspring were 0.604 and 0.396, respectively. The expected frequency distribution of the C-93 and T-93 alleles is the formula p2 + 2pq + q2 = 1.
Can be estimated using Allele frequency in pre-eclamptic offspring group is p = 0.5
With 37 and q = 0.463, the expected distribution of genotypes is C-93 / C.
-93 = 0.288, C-93 / T-93 = 0.502, T-93 / T-93 =
It should be 0.214. Allele frequency in control progeny group p = 0.6
With 04 and q = 0.396, the expected distribution of genotypes is C-93 / C.
-93 = 0.36, C-93 / T-93 = 0.478, T-93 / T-93 = 0
． Should be 156. The distribution of genotypes in the pre-eclamptic offspring group is significantly different compared to the control group (χ ² = 11.01, p <0.001, Table 1).

It was reported that no significant association was observed between the exon 8 deletion polymorphism of HLA-G and preeclamptic offspring (Humphrey et al., 1995). In this study, we genotyped preeclamptic and control progeny for the HLA-G deletion polymorphism. Preeclampsia group (n = 51) and control progeny group (n = 51)
= 55), a genetic analysis of HLA-G deletion polymorphisms showed that 12 (23.5%) and 13 (23.6%) homozygous for the normal allele, respectively.
), Homozygous for the deleted allele in 8 cases (15.7%) and 1 respectively.
Two cases (21.8%), as well as 31 cases (60.8%) and 30 cases (54.5%) of heterozygotes were shown.

The frequencies of normal and deletion alleles in preeclamptic offspring were 0.539 and 0.46, respectively. By comparison, the frequencies in control offspring were 0.509 and 0.491, respectively. In the pre-eclamptic progeny group, the expected distribution of genotypes is normal allele / normal allele = 0.291, normal allele / deletion allele = 0.497, deletion allele / deletion allele = It should be 0.212. In the control offspring group, the expected genotype distribution is
Normal allele / normal allele = 0.259, normal allele / deletion allele = 0.500, deletion allele / deletion allele = 0.241. The distribution of genotypes in the preeclamptic offspring group is not significantly different compared to the control group (χ ² = 0.69, p <0.30, Table 1).

Parents of preeclamptic and control progeny were genotyped for the C / T-93 genotype of HLA-G, and this genotype was analyzed along with the genotype of the progeny.
In this analysis, we found paternal C / T- that was not present in the maternal genotype.
The number of cases of offspring inheriting the 93 allele was evaluated. About the preeclamptic offspring,
41% of cases had a paternal allele of the C / T-93 genotype that was not present in the maternal genotype. In contrast, for control offspring, 28% of cases had paternal C / T-93 alleles not presented in the maternal genotype.

Discussion In this study, two common polymorphisms within the HLA-G gene in preeclamptic maternal and normal maternals were tested for association with preeclampsia. The two offspring come from southern Ireland and similar racial backgrounds.

The present invention is the first report to determine the association between maternal preeclampsia and fetal HLA-G genotype. Our results show a strong association between preeclampsia in maternal and heterozygosity for C / T-93 polymorphism in offspring. This indicates that transmission of the HLA-G allele to offspring is different in normal offspring than in preeclamptic offspring. This result indicates that screening for susceptibility to preeclampsia can be achieved by genotyping HLA-G in maternal and spouse. Furthermore, because preeclampsia is associated with intrauterine growth retardation and miscarriage, screening for susceptibility to intrauterine growth retardation, miscarriage and miscarriage-related infertility was also achieved by genotyping HLA-G in potential parents. It seems that it can be done.

Following these results, and to add further support for this important finding, we expanded the number of analytes analyzed. These subjects included further normal first trimester triads (mothers had no history of pregnancy-related problems), pregnant women with preeclampsia triads, and their mothers in the absence of pregnancy-related disorders including preeclampsia and miscarriage. A family (mother, father, first and second child) who has had two or more successful normal pregnancies in
, A family of mothers (mothers, fathers, who experienced preeclampsia in the first pregnancy and experienced a normal second pregnancy in the absence of pregnancy-related disorders including preeclampsia and abortion)
First and second child))). We have also included a recurrent couple cohort as preeclampsia has been shown to be associated with miscarriage. The C / T-93 polymorphism and the I / D-E8 polymorphism were genotyped in all individuals (Tables 2 and 6) and assigned halotypes to support our initial findings. And applied more detailed statistics.

HLA-G Linkage for Successful Pregnancy in Normal Pregnant Women For transmission analysis of polymorphisms in individuals, we applied a log-linear model and transmission imbalance test. We also tested other polymorphisms in the HLA-G gene. The frequencies of A / T-31 polymorphism, A / T-107 polymorphism and C / A-110 polymorphism in the sample cohorts were 1.0 / 0.0 and 0.9, respectively.
It was 5 / 0.05 and 0.94 / 0.06. For analysis, we still took advantage of the commonly occurring C / T-93 and I / D-E8 polymorphisms. Using an allele-specific amplification approach, we designated inherited and non-inherited alleles in the triad (Table 3). Since maternal, fetal genotypes (and especially fetal alleles of paternal origin) can potentially influence pregnancy outcome, a number of different comparisons were made using this data. The log-linear model of Weinberg et al. (1998) allows for an in-relationship scenario in which fetal genotype, parental genotype or combinations thereof are directly associated with risk. Case-Fit maximum likelihood log-linear of the triple data pair was fitted. This log-linear model has an expected number (for dual allele markers)
Predicted expectations for the 16 possible familial forms observed. This is due to several "genetic risk factors": (a) whether or not the offspring carried one or more copies of the allele, (b) the mother had one or more copies of the allele. Were carried, (c) maternal origin effects, and (d) paternal origin effects (Table 4). This analysis stratified the parental mating type assuming Hardy-Weinberg equilibrium. This corrects for the number of particular alleles found among the four parental alleles in a particular mating type. This is trivial, but has an unrelated effect on the allelic distribution among the offspring. The advantage of this framework is that nested models of different complexity start with a single allelic effect and are effectively compared by adding additional factors. We use a four factor model, followed by a gradual reduction model that automatically removes non-significant factors. Therefore, this provides a simpler model that accounts for the significant deviations from expectations.

The fit of the 4-parameter model to this data (Table 4) was significant for the C-93 allele (p = 0.006) and especially for the I-E8 allele (p =). 0.0001). Step removal of non-significant parameters reveals that most of this could be due to two effects: fetal HLA-G.
The effects due to alleles, and the strongest, are due to the effects of parental origin on each allele (Table 4). Thus, both C-93 and IE8 alleles are shown to be significantly lower among progeny, and when they occur, tend to be of paternal and maternal origin, respectively. The maternal allele itself becomes non-significantly biased once these other effects are considered. The I-E8 allele is
It appears to be more than four times more maternal in origin than expected (95% confidence interval 2.
2-9.8).

Highly significant differences were observed for C / T-93 and I / D-E8 alleles when maternal and paternal allele transmission frequencies were compared using a χ ² contingency table analysis. (P ₋₁ = 0.009 and p ₋₁ = 0.000001, respectively)
, Table 5, Table 3). This is maternal C-93, D-E8 and paternal T-93, IE
It reflected a deficiency in transmission of the 8 alleles (Table 3). Significant differences were also observed between the maternal and paternal untransmitted I / D-E8 alleles. This indicates that maternal genotype plays a role in pregnancy outcome.

We validated a very significant finding from log-linear modeling with simpler comparisons. The Transmission Disequilibrium Test evaluates whether to assess whether the transmission of maternal and paternal alleles from a heterozygous parent to offspring differs from the null expectation (at 50:50). And this is valid even when the Hardy-Weinberg equilibrium is disturbed by abnormal population structure. When the transmission disequilibrium test was applied, the transmission of C / T-93 and I / D-E8 alleles to offspring was not different from null expectations (Table 5). However, a significant difference from the null expectation was observed when maternal and paternal transmission frequencies were analyzed separately from each other.

Maternal T-93 to offspring (χ ² transfer imbalance test, p = 0.032) and IE
8 (χ ² transmission imbalance test, p = 0.005) and paternal DE
Transmission of the 8 (χ ² transmission imbalance test, p = 0.01) allele was also much more frequent than expected (Table 5). Maternal T-9 from a heterozygous mother to offspring
There is excessive transmission of the 3 allele. In particular, 30 of 44 descendants have C / T-
The maternal T-93 allele from the 93 heterozygous mother was inherited, and 40 offspring in 52 inherited the maternal I-E8 allele from the I / D-E8 heterozygous mother (Table 3). Furthermore, a contrasting over-transmission of the paternal D-E8 allele from the heterozygous father to the offspring (31 of 43 cases) was observed (Table 3).
. These findings are in good agreement with those of the log-linear model.

We tested this data to determine if transmission distortions could be due only to transmission to female or male offspring. However, in this case, 40 of the 52 offspring inherited the maternal I-E8 allele from the heterozygous mother, of which 19 were female and 21 were male, so this is the case. There was no evidence. Similarly, 31 of 43 offspring inherited the paternal D-E8 allele from the heterozygous father, of which 15
Were female and 16 were male.

The primary pregnant (primagravida) mothers investigated here had an I / D-E8 genotype frequency (observed genotype frequency: I / I; 17, I / D; 58, D / D;
15, for the expected I / I; 24, I / D; 45, D / D; 21), which was significantly different from the Hardy-Weinberg prediction (p ₁ = 0.006). The results of the transmission imbalance test show significant effects without assuming Hardy-Weinberg equilibrium, thus supporting the results of the log-linear model calculated assuming Hardy-Weinberg equilibrium.

The assignment of transmitted and untransmitted alleles to offspring was determined for five independent HLA-G polymorphisms, haplotype construction and comparison of transmitted and untransmitted haplotypes. Made possible. Thirteen haplotypes were observed (Table 6). Four of these, aa-a-a-b, a
-B-a-a-a, aa-a-a-a and ab-a-a-b were relatively common. Differences between the frequencies of maternal and paternal transmission were apparent for all four common haplotypes. This means that HLA-
We show that distortions in G allele transmission can still be attributed to biases in the transmission of C / T-93 and I / D-E8 polymorphisms. We then constructed haplotypes for the C / T-93 and I / D-E8 polymorphisms alone for comparison purposes.

The CD and TI haplotypes were the most common (Table 7).
The strength of linkage disequilibrium between the two markers is indicated by the high frequency of CD haplotypes. The imbalance for this (expressed as a ratio of maximum imbalance (D / Dmax)) is 0.344. Comparison of maternal and paternal transmission haplotypes to offspring using the χ ² contingency table analysis showed a highly significant difference between maternal and paternal transmission haplotypes to progeny (p ₃ = 0.000003). This is D
Both maternal haplotypes with the -E8 allele (CD and TD) and both paternal haplotypes with the I-E8 allele (CI and TI)
) Reflects a deficiency in transmission (Table 7). A significant difference was also observed between haplotypes that were not maternally transmitted to offspring and haplotypes that were not paternally transmitted (p ₃ = 0.028). This indicates that the maternal HLA-G genotype plays a role in pregnancy outcome.

Maternal and paternally transmitted haplotypes to individual offspring are shown in Table 8. A combination of maternally transmitted TI haplotype and paternally transmitted CD haplotype occurs in 21 (34%) of control offspring. In contrast, alternative possible combinations (maternally transmitted CD and paternally transmitted TI)
Does not occur in any of the control progeny, despite having made 22 crosses with this potential.

In first-trimester offspring, homozygosity did not deviate from Hardy-Weinberg expectations. Homozygous several pairs heterozygous observations number and the expected number of comparison in the progeny did not show any significant difference (p ₁ = 0.256 and p ₁ = 0.82 for the C / T-93). This indicates that opposition to homozygosity does not occur in the first trimester.

The significant distortion observed for transmission of the HLA-G allele to first-trimester offspring was due to maternal and paternal allele-specific HLA in normal first pregnancy.
-Provide evidence of fetal selection based on G. The observed choices are HLA-G
Most obvious was the I / D-E8 polymorphism. Log-linear models and transmission disequilibrium test analyzes show strong selection for maternal I-E8 and paternal D-E8 alleles in offspring. The effect of selection is most dramatic for the fetus of the I / D-E8 allele combination. In all samples, 37 of the 84 progeny have the maternal I-E8, paternal D allele combination. In contrast, there are only 5 progeny with the alternative combination of maternal D-E8, paternal I-E8 allele combinations (calculated from Table 8). These results indicate that maternal D-E8, paternal I-E8 fetuses undergo significantly increased post-zygotic prenatal loss, and HLA-G as a key gene affecting this process.
Is shown to be identified. Maternal D-E8 and paternal I-E8 offspring have approximately 29 defects.
%, And closely matches postnatal prenatal loss in expected mothers, which was estimated to occur at a frequency of about 31%, and in these cases, more than 20%, Losses occur very early in pregnancy and are clinically unrecognizable.

The maternal HLA-GI-E8 allele apparently confers a protective effect on the fetus, while the equivalent paternal allele has a dichotomous effect due to its deleterious effect, which is somewhat suggestive for imprinting the genome. However, the previous reports indicate that imprinting is HLA-G.
It shows that it does not occur at the locus. It is not clear why there are too many heterozygous mothers for the I / D-E8 genotype. I / D-E
An excess number of mothers heterozygous for the eight genotypes was found to be
Selected based on normal pregnancy outcomes, and all female populations were selected for infertile women (10
~ 15% frequency), women who have experienced miscarriage (10-15% frequency), or preeclampsia (
This represents a selected group of female populations, as it includes several other categories of women, including 5-10% frequency).

In the first-trimester offspring, homozygotes did not deviate from Hardy-Weinberg expectations. Comparison of the observed and predicted numbers of homozygous vs. heterozygous predicted in progeny showed no significant difference (p ₁ = 0 for C / T-93).
． About 45 and _{I / D-E8 p 1 =} 0.81). This indicates that selection for homozygotes does not occur at first pregnancy. Preferential transmission of maternal I-E8 and paternal D-E8 HLA-G allele pairs to offspring can be expected to result in increased heterozygosity in offspring. However, it appears that the selection of offspring is aimed at the combination of the maternal I-E8 and paternal D-E8 HLA-G alleles and against the combination of the paternal I-E8 and maternal D-E8 allele pairs. As such, the excess of the former heterozygote balances due to the lack of the latter.

This result indicates that the lineage imbalance occurs across the HLA locus, and thus the result is HLA-G.
And / or is proof that the HLA-G linked gene causes the selection effect observed here. C / T-93 polymorphism is a silent mutation, but I / D
-E8 polymorphism occurs in the 3'untranslated region (UTR) of genes. These polymorphisms are considered harmless. However, this evidence suggests that the deletion polymorphism
It shows that it has a functional effect on the HLA-G gene. The 14 bp sequence of the I / D-E8 polymorphism is found in primates and in the 3'UTR and / or HLA.
-Greatly conserved in the last intron of B, C, J, A and E. Polymorphic 1
Eleven of the 4 bp are repeated in the 7th intron of the HLA-G gene. The core sequence "atttgt" is repeated one or more times in the 3'UTR of class I genes, but the coding sequence is absent. I using the mfold program
Examination of the secondary structure around the / D-E8 polymorphism (Zuker, 1994) showed that the 14n sequence was contained in a region of the 3'UTR with extensive secondary structure, and this secondary structure was of the 14n sequence. It is shown to vary depending on the presence or absence (data not shown). Thus, the presence or absence of polymorphisms may affect stability and / or alternative splicing of HLA-G mRNA through the formation of alternative secondary structures.

Testing of human pre-transplant blastocysts showed that only 40% of the blastocysts express HLA-G, and such expression indicates cleavage rates in comparison to embryos lacking the HLA-G transcript. It was associated with an increase in Thus, polymorphisms that affect HLA-G expression appear to affect the rate of postnatal loss following zygosity by altering the rate of cleavage in the embryo.

Taken together, this result indicates that different HLA-G alleles and / or their combinations and / or alterations in HLA-G linked disequilibrium DNA in the fetus and / or fetal parent or parents are expressed as abortions. Providing evidence of causative prenatal loss after gaining zygosity or undetectable premature abortion manifested as unexplained infertility.

Identification of HLA-G as a Preeclampsia Gene A cohort of preeclamptic first-trimester triads (mother, father and first child) was identified at an obstetric hospital, sampled, and the HLA-G gene. Were genotyped for the following polymorphisms in: C / T at codon 93 (C / T-93) (Table 9).
, A / T at codon 107 (A / T 107), C / A at codon 110 (C /
A 110), and polymorphic insertions in the untranslated region of the exon 8 gene /
Deletion polymorphism (I / D-E8) (Table 9). Alleles transmitted and not transmitted to offspring were designated (Table 10 and Table 11).

HLA-G genotypes and haplotypes in preeclamptic triads were tested separately using transmission segregation analysis. The preeclamptic triad was also compared to the control first pregnancy triad cohort. A significant difference in C / T-93 allele frequency was observed between control and preeclamptic mothers (p ₁ = 0.03, Table 12).
. A significant difference in allele frequency of the I / D-E8 polymorphism was also observed between control and preeclamptic fathers (p ₁ = 0.02, Table 12). The frequency of 93-E8 haplotype, while (p ₃ = 0.03) between the control and pre-eclampsia maternal, between the control and preeclampsia father (p ₃ = 0.008, Table 12) were significantly different in the , And also significantly different between control and preeclamptic offspring (p ₃ = 0.03, Table 12).

The distribution of C / T-93 genotypes was significantly different between controls and preeclamptic triads (Table 12). Highly significant difference between control and preeclamptic offspring (
(p ₂ = 0.001), between control and preeclamptic father (p ₂ = 0.02), and between control and preeclamptic mother (p ₂ = 0.05). These differences are due to the preeclamptic offspring (p ₁ = 0.0002) and the preeclamptic father (p ₁ = 0.002).
0.021) significantly exceeded the Hardy-Weinberg equilibrium expectations, reflecting a significant excess of the C / T-93 heterozygotes, and significantly exceeded the Hardy-Weinberg expected I / D-E8 heterozygotes. The body was also observed in control mothers, preeclamptic offspring and preeclamptic fathers (Table 12).

An analysis of HLA-G haplotype sharing between offspring and mother was also performed. No significant differences between preeclamptic cases and controls were observed for fetal maternal sharing of the HLA-G allele or sharing of the paternally transmitted HLA-G allele. There were no significant differences observed in the sex of offspring between controls and cases of preeclampsia.

Comparison of maternal and paternally transmitted alleles and haplotype frequencies to control and preeclamptic offspring showed significant differences (Table 12). In particular, these differences were due to an excess maternally inherited T-93 / I-E8 haplotype and an excessive paternally inherited C-93 / D-E8 haplotype in the control offspring, as well as a defective maternally inherited C-93 / D-E8 haplotype. And defective paternally inherited T-93 / I-E8
Haplotypes were shown by comparison with preeclamptic offspring (Table 10). Furthermore, there were significant differences between the non-transmitted maternal I / D-E8 allele and the 93-E8 haplotype. This indicates that the maternal non-transmitted allele is associated with pregnancy outcome.

Only 12 of the 52 control progeny were heterozygous (I / DE).
8) We inherited the maternal D-E8 allele from the mother (Table 3). In contrast, the maternal D-E8 allele was transmitted to 21 of 36 preeclamptic offspring (Table 11). Taken together, these findings demonstrate a significant loss of maternal D-E8 transmission to control offspring and a contrasting excess of maternal D-E8 transmission to preeclamptic offspring.

Further analysis within the control and preeclamptic triad was achieved by comparison of maternal and paternally transmitted alleles and haplotypes to offspring. In control progeny, a highly significant difference was observed between transmission of maternal and paternal C / T-93 alleles (Table 5, p ₁ = 0.009, calculated from Table 3). This reflected a defect in the maternal C-93 and paternal T-93 transduction alleles (Table 3). Highly significant differences were also observed between transmission of maternal and paternal I / D-E8 alleles (Table 4, p ₁ = 0.000001, calculated from Table 3). This indicates a defect in transmission of maternal D-E8 and paternal I-E8 alleles to offspring (Table 3). A significant but contrasting difference between transmission of maternal and paternal alleles was present in preeclamptic offspring. Here, excessive transmission of maternal D-E8 and paternal I-E8 alleles,
Excessive transmission of maternal C-93 and paternal T-93 was observed (Table 13, Table 10).
. There was a significant difference between the maternally transmitted paternal haplotype and the paternally transmitted haplotype in both control and preeclamptic offspring (p ₃ = 0.0000, respectively).
03, Table 5 and p ₃ = 0.005). This is a defect in transmission of maternal C-93 / D-E8 and paternal T-93 / I-E8 haplotypes to control offspring, and maternal C-93 / D-E8 haplotypes and paternal to preeclamptic offspring. It shows an excess of T-93 / I-E8 haplotype transmission (Table 10).

Maternal and paternal transmission haplotypes to individual offspring are shown in Table 14. The combination of maternally transmitted C-93 / D-E8 haplotype and paternally transmitted T-93 / I-E8 haplotype occurs in 22 (35%) preeclamptic offspring, but not in any control offspring. In contrast, paternal C-93 / D-E8, maternal T-
The 93 / I-E8 haplotype combination occurs in both control and preeclamptic offspring, but is excess in control offspring. This finding is
It provides evidence that the HLA-G allele / haplotype combination in the fetus is the cause of preeclampsia in the mother.

Taken together, this result indicates that different fetal HLA-G alleles and / or combinations thereof, and / or mutations in DNA linkage disequilibrium with fetal HLA-G and / or fetal single parent or parents Provide evidence that it is the cause of preeclampsia.

Association of HLA-G with Recurrent Abortion A mother's cohort with three or more consecutive abortions was identified, sampled, and C / T-93 and I / in the HLA-G gene. Genotyped for the D-E8 polymorphism and reserved the 93-I-E8 haplotype. RSA mothers and RSA fathers were treated with C / T-93 and I / D-E8 allele frequencies, C / T
T-93 and I / D-E8 genotype distributions and 93-E8 haplotype frequencies were compared to a control and preeclamptic first pregnancy triad cohort. Table 16 shows the genotype and haplotype of the couple.

Significant differences in C / T-93 allele frequencies were observed between control and preeclamptic mothers (p ₁ = 0.03) and control and RSA mothers (p ₁ = 0.002).
, But not between preeclampsia and RSA mothers. 93-E
The frequency of the 8 haplotypes was higher between controls and preeclamptic mothers (p ₃ = 0.03).
), Significantly different between control and RSA mothers (p ₃ = 0.01),
There were no significant differences between preeclampsia and RSA mothers.

Mating partners for the female and male 93-E8 haplotypes were constructed. Fifty
% Of couples had maternally transmitted C-93 / D-E8 haplotypes and paternally transmitted T-93 / I
-Having fetal birth potential with the E8 haplotype combination. this is,
24% chance in control couples and 44% in preeclamptic couples
Compare with the possibility of. The chance of producing a fetus with the maternally transmitted D-E8 allele and the paternally transmitted I-E8 allele is 46% in the control couple,
70% in preeclamptic couples and 85% in consecutive miscarriages.
Maternally transmitted C-93 / D-E8 haplotypes and paternally transmitted T-93 / I in the fetus
The E8 haplotype combination is only found in preeclamptic offspring. A recurrent abortion couple (no. 15) must produce such offspring. Taken together, this result provides evidence that the fetal genotype associated with preeclampsia is also associated with miscarriage. Finally, females are homozygous for the T-93 / I-E8 haplotype and males are C-93 / D-E8 and T-93 /.
In the case of preeclampsia, the mating of couples with the I-E8 haplotype was 7 out of 63 individuals.
It was seen in the individual but not in the control. Two of the 20 recurrent miscarriage matings (no. 10 and 13 in Table 17) also had the same haplotype combinations. This indicates that females are homozygous for the T-93 / I-E8 haplotype and males are C-93 / D-E8 and T-93.
Partners with the / I-E8 haplotype provide evidence of susceptibility to preeclampsia and / or miscarriage. Taken together, this result provides evidence that miscarriage and preeclampsia are closely related and that miscarriage is a severe manifestation of PE. One offspring in the control triad of the T-93 / I-E8 and C-93 / D-E8 haplotype combination was that the mother was homozygous for T-93 / I-E8 and the father was C-93. 93 / D-E8 haplotype and C-93
/ I-E8 haplotype. Certain offspring in the control triad of T-93 / I-E8 and T-93 / I-E8 haplotype combinations were also found to be homozygous for the mother to T-93 / I-E8 and for the father to be , T-93 / I-E8 haplotypes and C-93 / I-E8 haplotypes. This result indicates that the non-transmitting male haplotype mainly affected pregnancy outcome, and that sperm / sperm contains factors that affect susceptibility to preeclampsia and miscarriage.

Taken together, this result indicates that different HLA-G alleles and / or combinations thereof and / or mutations in the HLA-G DNA linkage disequilibrium in the fetus, and / or the fetal single parent or parents cause abortion. Provide proof of

Induction of HLA-G Preeclampsia / Abortion Haplotype Resistance in First Pregnancy This result indicates that maternally transmitted D-E8 alleles and paternally transmitted I-E8 to offspring are associated with pregnancy outcomes. , And that the combination of the maternally transmitted C-93 / D-E8 haplotype and the paternally transmitted T-93 / I-E8 haplotype causes preeclampsia and abortion in the first pregnancy. We found that HL in 53 couples
AG transmission was analyzed. These couples had successfully had two pregnancies without experiencing a miscarriage or PE.

The chance of producing a fetus with the maternally transmitted D-E8 allele and the paternally transmitted I-E8 allele was 15% in this cohort of normal couples. Thus, this result is consistent with successful pregnancy outcomes and maternally transmitted D-E8 and paternally transmitted I-E8H.
It shows a clear correlation between the probability of having a fetus with the LA-G allele. Recurrent miscarriage is 85%, preeclampsia 70%, normal first pregnancy 46%, normal first and second pregnancy 15%.

The combination of the maternally transmitted C-93 / D-E8 haplotype and the paternally transmitted T-93 / I-E8 haplotype causes preeclampsia and abortion in the first pregnancy. Whether the normal pregnancy of the first pregnancy induces resistance to the pre-eclampsia / miscarriage haplotype combination when the preeclampsia and the miscarriage haplotype occur in the two pregnancies. It was determined. None of the firstborns were detected to have a combination of maternally transmitted C-93 / D-E8 haplotype and paternally transmitted T-93 / I-E8 haplotype. There were five second offspring with a combination of maternally transmitted C-93 / D-E8 haplotype and paternally transmitted T-93 / I-E8 haplotype (Table 18). Whether the preeclampsia pregnancies of the first trimester also induce resistance to the combined preeclampsia / miscarriage haplotype if preeclampsia and abortion haplotype occur in two pregnancies? It was determined. We analyzed 9 families whose mother had preeclampsia at the first pregnancy and had a normal second pregnancy. Maternal transmission C-93 / D-in the absence of preeclampsia, even though the same combination caused preeclampsia in the first pregnancy.
There were 2/3 progeny with a combination of E8 haplotype and paternally transmitted T-93 / I-E8 haplotype (Table 19).

This demonstrates that resistance to paternal antigen in the fetus is induced at first pregnancy. More specifically, resistance to the maternally transmitted C-93 / D-E8 haplotype in question and the paternally transmitted T-93 / I-E8 haplotype was induced in the first pregnancy. Thus, exposure to the HLA-G gene pair and / or combination thereof, and / or paternal antigens presented to the maternal immune system by HLA-G in the first pregnancy, results in resistance to preeclampsia / miscarriage haplotype combinations. And as a result, the haplotypes of these problems are 2
It can occur in the second pregnancy without associated preeclampsia or miscarriage.

The gene linkage association and correlation approach used in a cohort of multiple subjects provides evidence that HLA-G is a gene susceptible to normal pregnancy, preeclampsia and miscarriage. When preeclampsia is associated with intrauterine growth retardation, the HLA-G gene is also a susceptibility gene for intrauterine growth retardation. HLA-G is also a susceptibility gene for unexplained miscarriage-related infertility, when undetectable miscarriage often occurs too early. Exposure to fetal antigens including HLA-G in the first pregnancy has been shown to induce resistance to the antigen of interest in the first pregnancy, and thus preeclampsia, miscarriage, delayed in utero growth, miscarriage. It provides a means of potential treatment of infertility and autoimmune diseases associated with and a means of inducing resistance to foreign antigens for purposes such as transplantation of foreign tissue.

The HLA-G I / D-E8 polymorphism has been previously studied in pre-eclampsia, and no detectable relationship between susceptibility to pre-eclampsia and HLA-G was observed ( 24). This result is consistent with the result reported in the present invention that the effect of HLA-G is not seen when I / D-E8 polymorphism is analyzed independently in the related study alone.

[0133] The results of the present invention indicate that maternal and / or paternal genetic screening, and / or
Or show that fetal DNA is of value for predictive testing of susceptibility to preeclampsia, preeclampsia, intrauterine growth retardation, miscarriage and miscarriage-related infertility. further,
Transmission of the HLA-G allele to offspring in normal pregnancy differs from the normal expectation. Therefore, the results presented in the present invention show that genetic screening of maternal and / or paternal fetal DNA is valuable for predictive testing of susceptibility to normal pregnancy.

Preferably, fetal nucleic acid is isolated from any material containing nucleic acid of fetal origin in the mother, such as amniotic fluid, maternal blood, or chorionic villi. Furthermore, the results indicate that genetic screening of parents may also have value for predictive testing of susceptibility to preeclampsia.

The function of HLA-G is distinct from its role in regulating activation of NK cells, and the induction of IL-3 and IL-1β in PBMCs is not yet fully understood. , HLA-G is an excellent candidate for pre-eclampsia. This is because HLA-G is considered to play an important role in fetal-maternal immune interaction. C / T-93 HLA-G associated with preeclampsia
Alleles are silent polymorphisms, but their effects on HLA-G mRNA stability or splicing are unknown. This polymorphism and / or mutations associated with this polymorphism appear to play a causative role in preeclampsia.

In this study, we demonstrate a difference between pre-eclampsia and control progeny regarding the sharing of the paternal C / T-93 allele between the progeny and their mother. There is. This result indicates that preeclampsia can occur due to the presence of the HLA-G haplotype in the fetus not previously encountered by the mother. HLA-G is in a tight linkage disequilibrium at the HLA locus, so paternal HLA-G itself and / or
Or the presence of an extended paternal HLA haplotype in the fetus that was not previously encountered by the mother appears to cause preeclampsia. Furthermore, HLA-G is HL
Determining an extended paternal HLA haplotype and comparing haplotypes with a maternal HLA haplotype that are segregating in the fetus because they are in close linkage disequilibrium with the A locus allow diagnosis of susceptibility to preeclampsia .

Although other associations have been reported between preeclampsia and maternal genotype, the results reported in the present invention are more consistent with epidemiological studies on preeclampsia. In particular, the association between the fetal HLA-G genotype is: a) preeclampsia is more common in sisters than in normal populations, and b) preeclampsia is consistent in identical twin mothers. Not, and c) is consistent with the observation that preeclampsia can result from alteration of the male partner. Pre-eclampsia is rare in the second or subsequent pregnancies, indicating that initial exposure to functional HLA-G prevents pre-eclampsia. Furthermore, HLA-G is now known to induce the synthesis of IL-3 and IL-1β and downregulate the production of tumor necrosis factor α. These observations, combined with the results presented in the present invention, indicate that inhibitors of HLA-G protein, IL-3 and / or IL-1β, or tumor necrosis factor α are associated with intrauterine growth retardation, preeclampsia, abortion, And is useful for the treatment of miscarriage-related infertility.

The genotype of HLA-G associated with preeclampsia and abortion appears to have one of the following few results.

I) It may lead to a decrease in the expression of HLA-G, which is cellular and / or
Or reflected as a decrease in the level of soluble HLA-G (HLA-G primary transcripts are alternatively spliced to yield several different mRNAs. One of these alternatively spliced forms is: Includes intron 4. The open reading frame in this mRNA follows intron 4 and terminates 21 amino acids after the α3 domain, encoded by exon 4. Thus, the transmembrane region and HLA encoded by exon 5 -The cytoplasmic tail of G is eliminated, so the resulting protein is soluble.). Therefore,
By measuring the levels of cellular and / or soluble HLA-G and comparing these levels to the normally observed levels, it is possible to diagnose susceptibility to preeclampsia abortion.

Ii) HLA-G genotypes associated with preeclampsia can lead to variations in HLA-G mRNA and / or HLA-G protein. This can then be detected by characterization of HLA-G mRNA and / or protein. Thus, characterization of the HLA-G protein in pregnant, fetal and / or male mating partners of each makes it possible to diagnose susceptibility to preeclampsia and miscarriage.

Iii) HLA-G protein expression results directly or indirectly in changes in the levels of specific molecules such as IL-3, IL-1β and / or tumor necrosis factor α. The HLA-G genotype associated with preeclampsia can result in alterations in the expression of such molecules. Therefore, by measuring the levels of such molecules and comparing these levels to the normally observed levels, it is possible to diagnose susceptibility to preeclampsia and abortion.

Iv) The genotype of HLA-G associated with preeclampsia can result in decreased expression of HLA-G. This, in turn, results in increased trophoblast lysis by NK cells. Therefore, by measuring the levels of trophoblast-specific markers and comparing these levels to the normally observed levels, it is possible to diagnose susceptibility to preeclampsia and miscarriage.

Variants of HLA-G associated with preeclampsia and miscarriage as well as normal pregnancy appear to have one of a small number of the following results.

I) Variants may result in altered expression of the HLA-G splice form and its level reflected as altered levels of HLA-G splice form, including soluble HLA-G in serum. Therefore, by measuring the size, level and / or splice morphology of HLA-G mRNA and / or proteins, including levels of soluble HLA-G, and comparing these levels to normally observed levels, preeclampsia And susceptibility to miscarriage can be diagnosed.

Ii) Variants of HLA-G associated with preeclampsia and miscarriage can result in mutations in the HLA-G protein, which in turn can produce cellular and / or soluble H
It can be detected by protein characterization of LA-G. Thus, characterization of the HLA-G protein in pregnant, fetal and / or male mating partners of each makes it possible to diagnose susceptibility to preeclampsia and miscarriage.

Iii) HLA-G protein expression results directly or indirectly in changes in the levels of specific molecules such as IL-3, IL-1β and / or tumor necrosis factor α. HLA-G variants associated with preeclampsia can lead to altered expression of such molecules. Therefore, by measuring the levels of such molecules and comparing these levels to the normally observed levels, it is possible to diagnose susceptibility to preeclampsia and miscarriage.

Iv) Variants of HLA-G associated with preeclampsia and abortion may result in increased or decreased expression of paternal and / or maternal HLA-G. This in turn results in increased trophoblast lysis by NK cells and / or cytotoxic T cells. Thus, by measuring the levels of trophoblast-specific markers and comparing these levels to the normally observed levels, it is possible to diagnose susceptibility to preeclampsia.

V) HLA-G variants associated with preeclampsia and abortion may result in increased or decreased cell division rate in the embryo. Therefore, by measuring the cell division rate in cells expressing one or more HLA-G variants and any combination thereof,
It makes it possible to diagnose preeclampsia and susceptibility to miscarriage.

These results indicate that the HLA-G polymorphism plays a major role in the predisposition to normal preeclampsia and abortion outcomes in pregnancy, and that the effects of the parent haplotype combination and origin are HLA. -Shows that it mediates the effect of G polymorphisms on these outcomes. These results show that HLA-G between fetus and parent
Analysis of the results of heterozygous vs. homozygous mating showed a strong relationship between genotype and PE, and that transmission of the HLA-G allele to the preeclamptic but not control offspring was altered. Indicates that These results show that maternal HLA-G
Provided evidence for linkage of the I-E8 allele to normal pregnancy outcomes in primordial women, and maternal D-E8 allele and C to control offspring.
The observed defect in -93 / D-E8 haplotype transmission suggests a fetal selection based on the HLA-G genotype in normal pregnancies of primordial women. Motherhood D-
The altered transmission of the E8 allele to the fetus indicates that the effects seen in normal priminatal women are mediated by the maternal allele, which acts predominantly in the fetus. Thus, maternal HLA-G transmits a protective effect on the fetus that enhances the outcome of normal pregnancy. This finding indicates that maternal selection of HLA-GI-E8 and other protective HLA-G alleles occurs in normal pregnancies. In contrast, maternal D-E8
Since the allele was spread in heterozygous preeclamptic offspring, this indicates that susceptibility to preeclampsia occurs in part through the absence of the protective maternal HLA-G allele in the fetus. . Chi-square contingency table analysis is consistent with log-linear model analysis in that the C-93 allele was overexpressed in preeclamptic offspring, and a bias for I-E8 maternal inheritance was present in controls. In addition, a log-linear model showed that the fetal C-93 allele was under-represented in control offspring with a strong bias towards the paternal inheritance of that allele. This indicates that the paternal C-93 allele also either transmits protection or does not introduce a problematic effect on the fetus, which improves the likelihood of a normal pregnancy outcome. These results indicate that more than one-third cases of preeclampsia were maternal C-93 / D-, which were absent in controls.
Having an E8 paternal T-93 / I-E8 haplotype combination is in good agreement with the findings observed when the maternal and paternal haplotype combinations were constructed for individual control and preeclamptic offspring. To do. Taken together, this data shows a strong link between both maternal and paternal HLA-G alleles that act throughout the fetus and the outcome of normal pregnancy, and preeclampsia is associated with protective maternal and fetus-related effects in the fetus. It is shown to occur through the absence of protective or problematic paternal HLA-G alleles. Furthermore, it may be due to the presence of several functionally different HLA-G alleles, and more than one-third cases of preeclampsia may be due to specific maternal / paternal haplotype combinations Considering that,
These results indicate that HLA-G is highly effective in normal and preeclamptic pregnancies.

Alternatively, the protective fetal maternal HLA-G allele appears to occur through transmission of the maternal dominant allele to the fetus that is recognized as self by the maternal immune system. The protective fetal paternal allele appears to arise through the cross recognition of the paternal allele as self by the maternal immune system. The problematic fetal paternal allele appears to be generated by the maternal immune system through cross recognition of the paternal allele as non-self. This result indicates that maternal HLA-G during and / or before pregnancy.
And maternal education of the lymphocyte repertoire for and for paternal HLA-G during pregnancy. The results also indicate that certain paternal HLA-G alleles are compatible with the maternal immune system, while others are less compatible. The combination of less compatible / incompatible paternal HLA-G alleles with maternal alleles that do not protect against paternal alleles appears to result in susceptibility to preeclampsia and miscarriage.

The second offspring of a normal primigravida and a preeclamptic mother is maternal C-93 / D-E8 paternal T.
The fact that we have the -93 / I-E8 genotype in the absence of preeclampsia in the second pregnancy is due to the fact that maternal education for fetal paternal antigen occurs during the first birth and this education is HLA-G. It is evidence that it is mediated by. From this study,
It is clear that the polymorphisms analyzed and / or the closely linked polymorphisms in HLA-G or adjacent HLA genes enhance the outcome of normal pregnancy and directly contribute to the susceptibility to preeclampsia and abortion Is. One possible explanation is that the polymorphisms reported here destabilize HLA-G mRNA and / or alter the splicing and / or glycosylation patterns of HLA-G. possible. The presence or absence of polymorphisms appears to result in stability and / or alternative splicing of HLA-G mRNA. Thus, the protective fetal maternal HLA-G allele appears to result from the transmission of the maternal allele to the fetus, which may or may not be expressed in the embryo. The protective fetal paternal allele appears to result from the transmission of the paternal allele to the fetus, which may or may not be expressed in the embryo.

At least 12 different haplotypes have been described for the HLA-G gene. In view of the association observed between HLA-G and recurrent abortion, the combination of HLA-G alleles in the early fetus and / or HLA- in the mother.
The combination of G alleles generally appears to have a severe effect on the outcome of transplantation and appears to account for cases of infertility and miscarriage of unknown cause or idiopathic. The previously reported association between preeclampsia and intrauterine growth retardation appears to be linked to the effect of the parent, which is also the origin of the fetal HLA-G allele, and this Show that the maternal HLA-G allele also plays a role in fetal growth outcomes.

HLA-G may protect otherwise sensitive target cells from natural killer cell-mediated lysis via interaction with inhibitory receptors in natural killer cells. HLA-G may also stimulate an HLA-G restricted lymphocyte response. The HLA-G molecule can act as a target molecule in a lytic reaction with lymphocytes, and HLA-G is involved in the education of the lymphocyte repertoire. Therefore, pre-eclampsia, miscarriage, miscarriage-related infertility and intrauterine growth retardation appear to occur through mechanisms involving blood mononuclear cells such as natural killer cells and cytotoxic T lymphocytes. Here, the interaction between female mating partner T cells and fetal antigens is compromised compared to normal pregnancy. Therefore, attenuated interactions that lead to lack of tolerance result in cell killing. The attenuated interaction also results in a lack of stimulation of cells expressing the HLA-G molecule and / or H.
It can lead to a lack of stimulation of cells that interact with cells expressing the LA-G molecule. The fact that the maternal C-93 / D-E8 paternal T-93 / I-E8 HLA-G genotype can occur in the second pregnancies of cases of primordial preeclampsia without the symptoms of preeclampsia is: Education mediated by fetal HLA-G against fetal antigens occurs in the first pregnancy of such mothers, indicating that it overcomes the attenuated interactions in the second and subsequent pregnancies. The fact that the maternal C-93 / D-E8 genotype deletion and T-93 / I-E8 genotype excess is transmitted to control offspring but not to preeclamptic offspring is: We show that selection for a fetus expressing the mother's educated antigen occurs in normal pregnancy. The fact that preeclampsia rarely occurs in the second pregnancy when the first pregnancy was normal is due to the induction of education against fetal antigens mediated by HLA-G when the first pregnancy in a normal mother. During and / or before, and preeclampsia, miscarriage, miscarriage-related infertility, and intrauterine growth retardation, and / or lack of education for fetal antigens in female mating partners during and / or before pregnancy. Show that it results from improper induction of education. HL in the fetus
Lack of education and / or attenuated induction of paternal antigens, such as defective HLA-G interactions with A-G and / or natural killer cells, may result in lack of trophoblast lysis and / or trophoblast stimulation. Which can result in reduced trophoblast function and / or lack of stimulation of cells that interact with the trophoblast. Therefore, H
Conditions linked to LA-G (eg, preeclampsia, miscarriage, miscarriage-related infertility and intrauterine growth retardation) are associated with blood mononuclear cell-mediated killing and / or monotherapy of accessible fetal tissues such as trophoblasts. Lack of trophoblast stimulation due to attenuated HLA-G interaction with nuclear trophoblasts and / or attenuated HLA with trophoblasts
It appears to occur through the lack of stimulation of blood mononuclear cells due to the -G interaction. Major histocompatibility (MHC) molecules (eg, HLA-G) interact with blood mononuclear cells, including cytotoxic T cells and natural killer cells, and thus, compared to normal pregnancy, maternal blood Abnormal interactions between nuclear cells and fetal cells presenting MHC / MHC antigen complexes and / or MHC / MHC antigen complexes secreted by fetal cells during preeclampsia, abortion and intrauterine growth retardation It seems to exist. Thus, blood mononuclear cell responses and / or trophoblast responses to such interactions appear to be aberrant in HLA-G related disorders. In particular, the cytokine response produced as a result of such interactions appears to be abnormal compared to normal conditions.

Therefore, diagnosis of susceptibility to preeclampsia, miscarriage, miscarriage-related infertility and in utero growth retardation and prediction of outcome of pregnancy are both direct and indirect measures of education for fetal antigens in female mating partners and / or Or blood mononuclear cells and H
It can be achieved by direct and indirect measurement of the interaction with cells expressing LA-G and / or HLA-G. Furthermore, direct and indirect measures of education for fetal antigens in female mating partners, and / or direct and indirect measurements of natural killer cell activity for HLA-G expressing cells and / or blood in partners mating with females. Direct and indirect measurement of the interaction between mononuclear cells and HLA-G and / or HLA-G expressing cells provides a means to monitor the progress of pregnancy.

Induction of education for fetal antigens in female mating partners by treatment with HLA-G and / or peptides known to bind to HLA-G is associated with preeclampsia, miscarriage, miscarriage-related infertility and Intrauterine growth retardation as well as any other HL
It constitutes a therapeutic means for the prevention and / or treatment of AG-related disorders.

The finding that the combination of HLA-G variants in the fetus is closely associated with preeclampsia and abortion, combined with the fact that HLA-G interacts with blood mononuclear cells. Further means for preventing and / or treating preeclampsia, miscarriage, miscarriage-related infertility and intrauterine growth retardation by inhibiting and / or altering the interaction of HLA-G variants with blood mononuclear cells. I will provide a. This can be achieved by treatment with HLA-G and / or variants of HLA-G and / or one or more molecules that recognize one or more HLA-G receptors on blood mononuclear cells, and / or HLA-. G gene and / or HLA-G gene variants and / or can be achieved by any one or a combination of approaches including inactivation of one or more HLA-G receptors on blood mononuclear cells. For example, this may be treatment with HLA-G-specific and / or HLA-G receptor-specific antibodies that interfere with HLA-G-blood mononuclear cell interactions and / or HLA-G and / or HLA-G on blood mononuclear cells. / Or treatment with one or more enzymes that recognize and alter HLA-G receptors and / or one or more peptides that bind to HLA-G and / or HLA-G receptors on blood mononuclear cells Achieved by the treatment Alternatively, the inhibition of the interaction of one or more HLA-G variants with blood mononuclear cells may be achieved by the HLA-G gene or HLA-G on blood mononuclear cells.
This can be achieved by inactivating genetic variants and / or one or more HLA-G receptors. This can be achieved through one or more gene inactivation approaches (eg,
With one or more nucleic acid antisense and / or ribozyme molecules that inhibit the expression of HLA-G genes and / or HLA-G gene variants and / or one or more HLA-G receptors on blood mononuclear cells Treatment). It has also been found through the use of gene therapy approaches to HLA-in somatically or in the germ line at one or both partners of a mating mating.
It can be achieved by inactivating the G gene, thereby introducing a molecule based on an inhibitory nucleic acid (eg, antisense and / or ribozyme) into the individual. It also transfers all or part of the HLA-G gene to the endogenous H
Inactivating the HLA-G gene in one or both partners of a mating mating couple, somatically or in the germ line, through recombination with LA-G and introduction in a manner that inactivates it Can be achieved by Alternatively, in the method of gene therapy of any of the HLA-G gene and / or a variant of the HLA-G gene and / or its receptor, the expression product amount of such gene in the individual is increased to obtain HLA-G in the individual. And / or may be used to fill in any deficiencies in its receptor. Therefore, modifying the interaction of HLA-G and / or HLA-G variants with blood mononuclear cells may involve introducing one or more HLA-G gene variants into somatic cells and / or mating. It can be achieved by introducing into one or both of the germ lines of the married couple or into the fertilized egg or cells arising from the fertilized egg prior to transplantation. This is especially important for increasing fertility for animal breeding purposes. For example, introduction of one or more HLA-G gene variants into the mating line of one or both mating mates, or into the fertilized egg or cells resulting from the fertilized egg (wherein
HLA-G variants are compatible with future mothers) provide a means of improving fertility and pregnancy outcomes resulting from any incompatibility between fetal HLA-G and maternal cells in the mother. .

HLA-G binds to a diverse but limited number of peptides in a manner similar to that found for classical class I molecules, and HLA-G is expressed in human thymus. , Which raises the possibility that maternal non-responsiveness to HLA-G expressing fetal tissues can be determined in the thymus by central presentation of this MHC molecule in the medullary epithelium (Crisa et al., 1997).
). It is known that HLA-G can stimulate the HLA-G restricted cytotoxic T lymphocyte response, and the HLA-G molecule can act as a target molecule in the lytic reaction on cytotoxic T lymphocytes. , And in vivo expressed in transgenic animals, HLA-G is involved in the education of the lymphocyte repertoire (S
chmidt et al., 1997). The present invention shows that the induction of education against fetal antigens occurs during pregnancy and results from exposure of the mother to fetal antigens during pregnancy.
A combination of HLA-G alleles unacceptable in the first pregnancy and / or linked to preeclampsia was acceptable in the second pregnancy without any associated pregnancy complications. . Therefore, the induction of education for fetal antigens is
It appears to result from a process involving LA-G. Thus, the present invention mimics the exposure to fetal antigens that occurs during pregnancy to allow HLA-G in individuals.
And / or provide a means of inducing education that includes tolerance to peptides bound to HLA-G. Thus, treatment of an individual with HLA-G and / or peptides known to bind to HLA-G constitutes a means of inducing education in the individual against the antigen. In particular, it provides a means of inducing susceptibility to pre-eclampsia, susceptibility to miscarriage, autoimmune disease and tolerance to antigens resulting in transplant rejection.

In normal pregnancy, direct and indirect of the levels and / or activities of molecules resulting from the interaction of fetal cells expressing HLA-G with blood mononuclear cells (eg lymphocytes and natural killer cells). The modification allows the pregnancy to progress properly. HLA-G and / or HL in preeclampsia, miscarriage, miscarriage-related infertility and intrauterine growth retardation and any other HLA-G related disorders
Changes in the level and / or activity of molecules that result from the interaction of AG-expressing fetal cells with blood mononuclear cells (eg, lymphocytes and natural killer cells) are compared to those that occur during normal pregnancy. It seems that it has been weakened. Therefore, mimicking altered levels and / or activity of one or more molecules resulting from the interaction between HLA-G and / or HLA-G expressing fetal cells and blood mononuclear cells in an individual is preeclamptic. Constitutes a therapeutic means for the prevention and / or treatment of illness, miscarriage, miscarriage-related infertility and intrauterine growth retardation and any other HLA-G related disorders.

Maternal HL transmitted to control offspring but not to preeclamptic offspring
Deletion of the A-G C-93 / D-E8 genotype and excess of the T-93 / I-E8 genotype suggest that fetal selection in normal pregnancy depends on the HLA-G genotype. For the purposes of fertility, and in particular for in vitro fertilization and embryo transfer in animals, HLA-G and HLA-G in men and / or women associated with successful normal first pregnancy with a particular mating partner. By selecting one or both mating partners, sperm and / or egg donors and / or embryo recipients based on HLA-G homolog genotype and / or serotype and / or activity, fertility and It provides a means of improving the success rate of in vitro fertilization as well as embryo transfer in animals and improving the outcome of pregnancy.

HLA-G protects trophoblasts from blood mononuclear cell-mediated killing, so that direct and indirect measurements of measurable substances derived from trophoblast cell killing can lead to preeclampsia, abortion, It should be possible to diagnose susceptibility to intrauterine growth retardation and to monitor pregnancy in humans and animals for normal progression and progression to preeclampsia, miscarriage and intrauterine growth retardation. More particularly, the interaction between MHC molecules (eg HLA-G) and blood mononuclear cells is known to directly and indirectly alter the synthesis and levels of some cytokines. Similarly, trophoblasts are known to synthesize and secrete several cytokines. In particular, altered regulation of some of these cytokines is predicted to attenuate fetal-maternal immune interactions, and pre-eclampsia and / or eclampsia and / or intrauterine growth retardation and / or abortion and / or It can manifest as miscarriage-related infertility. For example, the interaction between HLA-G expressing cells and blood mononuclear cells results in interleukin 3 (IL-3) and interleukin 1 from blood mononuclear cells.
Increases the amount of β (IL-1β) and decreases the amount of tumor necrosis factor α (TNF-α) release. Trophoblasts are known to produce the immunosuppressive cytokine interleukin 10, which is a cytokine that strongly inhibits allogeneic responses in mixed lymphocyte reactions. Trophoblasts are also interleukins 2, which are cytokines that both protect the fetus and protect the invading trophoblasts by participating in the activation of maternal killing cells. And its receptor, which plays a role in the regulation of umbilical blood flow mediated through induction of cyclooxygenase-2, which is interleukin-4.
Produce interleukin 6, which appears to play a role in tissue remodeling associated with placenta formation, suggesting a role in vascular tone and in regulating blood flow during pregnancy. Is known.

Since there are indications that pre-eclampsia, miscarriage, miscarriage-related infertility and in utero growth retardation occur through HLA-G mediated mechanisms, we screen for agents that could potentially be used as diagnostic indicators and therapeutics. There are some obvious ways to do this. Screening of gene expression profiles using a DNA probe array reveals genes expressed in HLA-G expressing cells and blood mononuclear cells as well as genes whose expression changes as a result of interaction between HLA-G and blood mononuclear cells. Allows identification. Gene expression profile in HLA-G expressing cells and / or blood mononuclear cells and / or HLA-G expressing cells interacting with blood mononuclear cells and / or blood mononuclear cells interacting with HLA-G Comparison allows identification of agents that could potentially be used as diagnostic indicators and therapeutic agents for pre-eclampsia, miscarriage, miscarriage-related infertility and intrauterine growth retardation.

HLA-G function and HLA-G expression can be measured. Therefore, HLA-G
For agents that alter the expression and / or function of and / or mimic that function for screening for potential therapeutic agents for preeclampsia, miscarriage, miscarriage-related infertility, and intrauterine growth retardation. To provide a method.

The terms “comprising / including” and “having / including” as used herein with reference to the present invention, identify the presence of a feature, integer, step or ingredient referred to. , But does not exclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0164]   (References)

[0165]

[Equation 1]

[0166]

[Equation 2]

[0167]

[Equation 3] (Table 1: HLA-GC in preeclamptic and control offspring.
/ T-93 (C1488T) polymorphism and I / D-E8 (exon deletion) polymorphism genotype and allele distribution)

[0168]

[Table 1] (Table 2: HLA-G C / T-93 polymorphisms and I / in the first trimester triad)
D-E8 polymorphism genotype distribution and allele distribution)

[0169]

[Table 2] (Table 3: Results of genotypic crosses for HLA-G polymorphisms in control triads and preeclamptic triads).

[0170]

[Table 3] (Table 4: Relative risk of fetal, maternal and paternal lineage effects in a log-linear model).

[0171]

[Table 4] (Table 5: Comparison of the first pregnancy within the triad)

[0172]

[Table 5] (Table 6: Extended haplotype transmission and frequency)

[0173]

[Table 6] (Table 7: Transmitted HLA-G haplotypes and non-transmitted HLA-G haplotypes to offspring in first trimester triads).

[0174]

[Table 7] (Table 8: Transmitted HLA-G haplotypes and non-transmitted HLA-G in triplicate.
Haplotype)

[0175]

[Table 8] (Table 9: Genotype and allele distribution of HLA-G C / T-93 polymorphism and I / D-E8 polymorphism in the triad of preeclamptic first trimesters)

[0176]

[Table 9] (Table 10: Transmitted HLA-G haplotypes and untransmitted HLA-G haplotypes to offspring in control and pre-eclamptic triads).

[0177]

[Table 10] (Table 11: HLA-G in control and preeclamptic tribes.
Results of genotypic crosses for polymorphisms)

[0178]

[Table 11] (Table 12: Comparison between control triad and preeclamptic triad).

[0179]

[Table 12] (Table 13: Comparison within control triad and pre-eclampsia triad).

[0180]

[Table 13] (Table 14: Transmission HLA in control and preeclamptic tribes.
-G haplotype and non-transmitted HLA-G haplotype)

[0181]

[Table 14] (Table 15: Relative risk of fetal, maternal and paternal origin effects in a log-linear model)

[0182]

[Table 15] (Table 16: HLA-G C / T-93 polymorphisms and I in recurrent abortion couples)
/ D-E8 polymorphism genotype distribution and allele distribution)

[0183]

[Table 16] (Table 17: HLA-G haplotypes in recurrent miscarriages)

[0184]

[Table 17] Table 18: Transmitted and non-transmitted HLA-G haplotypes (expanded genotype) in first and second offspring of normal mothers.

[0185]

[Table 18] (Table 19: Transmission H in the first and second offspring of mothers with preeclampsia in their first pregnancy)
Expanded genotype of LA-G and non-transmitted HLA-G)

[0186]

[Table 19]

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 15/00 A61P 15/08 4C085 15/06 C12Q 1/02 4C087 15/08 1/68 C12Q 1/02 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 33/564 Z 33/50 33/68 33/564 C12N 15/00 ZNAA 33/68 A61K 37/02 // C12N 5/10 C12N 5 / 00 B (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG) , ZW), E A (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ , DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Kwan, Kathleen A .. County of Ireland County Cork, Kiravalen, Monanimi Upper (No Address) (72) Inventor Jenkins, David M. Cork, Ireland, Passage West, The Highlands 27 (72) Inventor McCarthy, Tommy Buoy. Ireland Cork, Montenotte, Vistavilla (No house number) F-term (reference) 2G045 AA27 BA20 BB20 CA17 CA25 CB01 CB14 DA12 DA13 DA14 DA36 DA80 FB02 FB03 FB05 JA01 JA09 JA20 4B024 AA01 AA11 BA80 CA01 CA04 CA06 CA11 CA14 CA12 CA14 CA14 CA12 CA14 CA12 CA14 QA01 QA07 QA12 QA13 QA18 QA19 QQ03 QQ42 QQ52 QR15 QR32 QR35 QR56 QR62 QS03 QS10 QS16 QS25 QS34 QS36 QX07 4B065 AA94X CA24 CA46 4C084 AA02 AA4A01 A14 A14 A1 4A1 A14 A1 4A1 A14 A1 4A1 A14 A15 A01 NA14 DA15 MA16 NA16 DA18 DA14 MA15 DA16 DA18

Claims (26)

[Claims] 1. A method of diagnosing susceptibility to normal pregnancy, preeclampsia and / or eclampsia, and / or intrauterine growth retardation, and / or abortion and / or abortion-related infertility. Has the following steps: a) obtaining a body fluid and / or tissue sample from a female and / or a male and / or a fetus; and b) including all or part of the HLA-G nucleic acid and / or the HLA-G linked nucleic acid. Or c) detecting variant forms of all or part of the HLA-G protein and / or the protein encoded by the HLA-G linked gene; or d) encoded by HLA-G. Protein and / or all or one of the proteins encoded by the HLA-G linked genes Measuring the functional activity of the region; or e) the mRN transcribed from the HLA-G mRNA or HLA-G linked gene.
Measuring the size and / or level of all or part of A; or f) the size and / or size of all or part of the HLA-G protein and / or the protein encoded by the HLA-G linked gene. Either measuring the levels; or g) quantifying the cells or molecules whose concentrations change as a result of the HLA-G action; and h) normal pregnancy and / or preeclampsia and / or Female and / or male and / or fetal parameters of pregnancy with eclampsia and / or intrauterine growth retardation, and / or susceptibility to the consequences of miscarriage and / or the consequences of miscarriage and / or b) to g). Comparing the parameters of the two. 2. The HLA-G nucleic acid comprises the presence of the C and / or T allele of codon 93 in exon 3 and / or the insertion and / or of exon 8.
Or the method of claim 1, wherein the method is analyzed for deletion alleles. 3. One or more of the HLAs for functional activity of the HLA-G.
-G sequence variants, and / or said HLA-G mRNA and / or
2. The method of claim 1, wherein the effect of one or more of the HLA-G sequence variants on the size and / or level of all or part of the encoded polypeptide of HLA-G mRNA is measured. 4. All or part of any of the HLA-G and / or HLA-G linked sequences is preferably polymerase chain reaction, nucleic acid sequence based amplification, self-sustaining sequence replication, transcription mediated amplification, 3. The method of claim 1 or 2, which is amplified by a method or combination of methods selected from strand displacement amplification and ligase chain reaction. 5. The method according to claims 1-4, wherein the comparison of the identified one or more variants is carried out by association and / or linkage analysis and / or transmission analysis. 6. The method according to claim 1, wherein all or part of the HLA-G sequence is cloned into a vector. 7. The method according to any of claims 1 to 6, wherein all or part of the nucleic acid sequence is DNA sequenced, glycosylase mediated polymorphism detection,
Restriction fragment length polymorphism analysis, enzymatic or chemical cleavage analysis, DNA and / or RNA probes and / or DNA probe arrays, and / or hybridization to allele-specific DNA probes and / or allele-specific RNA probes, A method identified by a method or combination of methods selected from allele-specific amplification analysis, electrophoretic mobility analysis, and 5'nuclease assay analysis. 8. All or part of HLA-G and / or all or part of one or more variants thereof as a polypeptide in vitro and / or in prokaryotic and / or eukaryotic cells. 8. The method of any of claims 1-7, which is expressed. 9. The method according to claim 1, wherein the cells in step (g) are blood mononuclear cells and / or their T cell subsets and / or natural killer cell subsets, and / or HLA-G expressing cells. Method. 10. A method according to any one of claims 1 to 9, wherein H
Any combination of LA-G and / or variants thereof, and / or blood mononuclear cells, and / or activity of a subset of such cells selected from T cells and / or natural killer cells has one activity The following procedures above: a) The interaction of HLA-G and / or variants thereof with blood mononuclear cells and / or a subset thereof is described below with respect to HLA-G expressing cells and / or blood mononuclear cells. A procedure for measuring by assessing one or more of: cell proliferation, transformation, cytotoxic response, surface marker expression, cytokine production, conjugate formation and target specificity, (b) HLA-G mRNA and / or its A procedure for determining the size and / or level of all or part of the encoded polypeptide, (c) Procedure for measuring the peptide binding ability of all or part of HLA-G and / or its variants, (d) HL of all or part of HLA-G and / or their variants
Procedure for measuring the ability to bind to the A-G receptor, (e) its level altered as a result of the interaction of HLA-G and / or their variants and / or cell-expressed HLA-G with blood mononuclear cells. And / or (f) measuring the expression level of one or more genes and / or proteins in HLA-G expressing cells. 11. A method according to any one of claims 1 to 10, wherein blood mononuclear cells and / or a subset thereof, and / or HL.
Cells expressing all or part of A-G and / or their HLA-G linked variants, and / or variants that completely and / or partially match female and / or male and / or fetal The method selected from the test panel. 12. The method of any of claims 1-11, wherein the HLA-G is partially or completely purified from cells expressing HLA-G. 13. HLA-G is detected by immunoassay with one or more antibodies specific for HLA-G and / or variants thereof.
The method described in any one of. 14. The HLA-A gene, HLA-B gene, HLA-C gene, HLA-E gene, HLA-F gene and all or part of the HLA-H gene are female and / or male and / or fetus. 14. The method according to any of claims 1-13, which is analyzed in. 15. The molecule of step (g) is IL-1β, IL-2, IL-3, I.
A trophoblast-specific marker selected from L-4, IL-6, IL-10 and tumor necrosis factor α or selected from cytokeratin, pregnancy-specific glycoprotein I, human chorionic gonadotropin and human placental lactogen The method of claim 1, wherein: 16. A method for screening for a drug that can potentially be used as a diagnostic indicator and / or a diagnostic drug targeting preeclampsia, miscarriage, miscarriage-related infertility and intrauterine growth retardation by the following steps: Method: a) Interaction of HLA-G and / or HLA-G expressing cells in HLA-G expressing cells and / or blood mononuclear cells and / or their T cell subsets and / or natural killer cell subsets. A subsequent step of measuring the expression level of one or more genes and / or proteins, b) the expression level identified in step a) and a normal and / or preeclamptic and / or intrauterine growth retarded pregnancy And / or abortion pregnancy and / or
Or in abortion-related infertility, HLA-G expressing cells and / or blood mononuclear cells after interaction with HLA-G and / or HLA-G expressing cells, and / or their T cell subsets and / or natural Comparing the expression level in the killer cell subset. 17. The method according to claim 10 or 16, wherein the gene expression and / or protein expression is cDNA and / or R derived from a cell.
Hybridization between NA and DNA probe and / or RNA probe and / or nucleic acid probe array, quantitative amplification method, reverse transcriptase-
Polymerase chain reaction (RT-PCR), 5'nuclease assay, ribonuclease protection assay and S1 nuclease assay, one-dimensional and / or two-dimensional gel electrophoresis and protein staining, enzyme linked immunosorbent assay (ELISA), radioimmunoassay ( RIA), protein truncation test (PTT), immunoradiometric assay (IRMA), and immunoenzymatic test (IEMA), sandwich assay with monoclonal and / or polyclonal antibodies and one or more using Western blotting The method of being determined by any one method selected from the detection of proteins, or a combination method. 18. A pharmaceutically effective amount of HLA-G and / or HLA.
-G expressing cells and / or one or more peptides that bind to HLA-G, H
Blood mononuclear cells from donors and / or test panels known to interact with LA-G variants, cytokines and IL-1β, IL-2, IL-
3, IL-4, IL-6, IL-10 and any combination thereof including tumor necrosis factor alpha and / or an inhibitor of cytokine and / or an inhibitor of tumor necrosis factor alpha and / or a derivative of cytokine and / or Alternatively, a pharmaceutical composition comprising a derivative of tumor necrosis factor α, optionally together with a pharmaceutically acceptable carrier or excipient. 19. A HLA-G or HLA in a method of screening for a potential therapeutic agent for the treatment of a condition selected from pre-eclampsia, eclampsia, intrauterine growth retardation, miscarriage and susceptibility to miscarriage-related infertility. -Use of G-expressed genes. 20. A method of preventing susceptibility to preeclampsia and / or eclampsia, and / or intrauterine growth retardation, and / or miscarriage and / or miscarriage-related infertility selected from the following: a) pharmaceutically effective Treatment of a female with all or part of HLA-G and / or a peptide that binds HLA-G and / or HLA-G expressing cells; b) a pharmaceutically effective amount of the molecule, or its level or Activity is HLA-G
Treatment of women with all or part of an inhibitor of a molecule that is altered directly or indirectly by an action; c) a pharmaceutical that inhibits the interaction between HLA-G and one or more of its receptors. Treatment of the female with all or part of the therapeutically effective amount of the molecule; d) treatment of the female with all or part of the pharmaceutically effective amount of the agent that alters HLA-G expression; e) HLA. -Treatment of a woman with a pharmaceutically effective amount of all or part of a drug that alters G-related blood mononuclear cell activity; f) pharmaceutically effective, which mimics all or part of the HLA-G action. Treatment of the female with all or part of a different amount of the drug; g) treatment of the female with blood mononuclear cells that recognize fetal HLA-G and / or autologous HLA-G; h) HLA-G and / or HLA. -G expressing cells or the Treatment of women in variant; i) HLA-G and / or HLA-G expressing cells and / or HLA
Treatment of women with one or more antibodies that bind to any receptor for G; j) introduction of one or more variants of the HLA-G gene and / or its receptors into women and / or men; k). Introduction of an inhibitor of HLA-G gene expression and / or its receptor into women and / or men; l) one or more variants of the HLA-G gene and / or lack of its receptor in women and / or men. activation. 21. A method for improving the success of pregnancy: a) sperm from a male with a different HLA-G genotype prior to mating with a male with a different HLA-G genotype. Known in vitro fertilization prior to in vitro fertilization used and / or embryo transfer in which the male HLA-G is a different HLA-G genotype
-Pretreatment of a woman with sperm from a male having a G genotype and / or an attenuated form thereof and / or semen and / or a fraction thereof; b) prior to in vitro fertilization a known HLA -G genotype sperm and HLA-G different
A step of mixing male with sperm from the genotype and / or an attenuated form thereof, and / or semen and / or a fraction thereof. 22. The method according to claim 21, wherein the results of fertilization and / or pregnancy are male and / or female partners, and / or sperm and / or eggs, and / or fertilized eggs and / or Or improved by selection of recipients of zygotes and / or embryos, so that (a) their HLA-
The G and / or HLA genotype and / or serotype, or (b) the activity of blood mononuclear cells interacting with their HLA-G and / or HLA-G is indicative of normal pregnancy results, and A method that is not associated with susceptibility to pre-eclampsia and / or eclampsia, and / or intrauterine growth retardation, and / or miscarriage and / or miscarriage-related infertility. 23. To diagnose susceptibility to normal pregnancy, preeclampsia and / or eclampsia, and / or intrauterine growth retardation, and / or susceptibility to miscarriage and / or miscarriage-related infertility, or to the progress of pregnancy. A kit for monitoring, which kit comprises: a) an oligonucleotide primer for amplifying all or part of HLA-G gene and / or HLA-G linked DNA; b) DNA / RNA polymerase, reverse Copy enzyme, deoxyribonucleotide d
Amplification reagents for amplification of genomic DNA and / or RNA segments selected from ATP, dCTP, dGTP, dTTP and dUTP, and / or ribonucleotide ATP, CTP, GTP, TTP and UTP, and reaction buffers; c ) Reagents for identifying sequence variants in DNA and / or RNA;
d) A kit containing control DNA and / or RNA. 24. Use of a DNA sequence selected from any one of SEQ ID NOS: 1-21, comprising: susceptibility to normal pregnancy, preeclampsia and / or eclampsia, and / or intrauterine growth retardation, and Screening for potential therapeutic agents for diagnosing susceptibility to abortion and / or abortion-related infertility, or in a test kit for diagnosing, for monitoring the progress of pregnancy, for use in the manufacture of a medicament In a method of screening for potential diagnostic indicators and / or drug targets, in a method of improving the success of pregnancy, or in preeclampsia and / or eclampsia, and / or in utero growth retardation, and / or miscarriage and / or Or how to prevent susceptibility to miscarriage-related infertility In order to monitor the progress of pregnancy, DN
Use of A array. 25. A method of inducing tolerance to non-self tissue in a host, which method comprises deriving from HLA-G and / or HLA-G and / or non-self tissue loaded with a peptide derived from non-self tissue. Or related HLA-G
Administering expressing cells, and / or non-self tissue carrying HLA-G introduced so that HLA-G is expressed in all or part of said tissue,
Method. 26. A method of treating an autoimmune disease, which method comprises HLA-G.
And / or HLA-G loaded with peptides derived from self and / or non-self tissue and / or specific autoimmune antigen, and / or HLA-G expressing cells derived from self and / or non-self tissue, and / or Or HLA
-A method comprising the step of administering self- and / or non-self tissue carrying an HLA-G gene introduced such that G is expressed in all or part of said tissue.