WO2020076065A1

WO2020076065A1 - Biomarker composition for diagnosing massive perivillous fibrin deposition associated with miscarriage, and use thereof

Info

Publication number: WO2020076065A1
Application number: PCT/KR2019/013225
Authority: WO
Inventors: 김종재; 김은나; 심재윤; 김경곤; 유지영; 이중엽; 황도영
Original assignee: 울산대학교 산학협력단; 재단법인 아산사회복지재단
Priority date: 2018-10-08
Filing date: 2019-10-08
Publication date: 2020-04-16
Also published as: KR102333647B1; KR20210111195A

Abstract

The present invention pertains to a biomarker composition for diagnosing massive perivillous fibrin deposition (MPFD) associated with miscarriage and a use thereof. A protein contained as an active ingredient in the biomarker composition according to the present invention for diagnosing massive perivillous fibrin deposition associated with miscarriage is specifically found only in maternal blood diagnosed with fibrin-type MPFD (f-MPFD), and can thus be useful for diagnosis with f-MPFD and prevent in advance habitual miscarriage caused by f-MPFD. Moreover, the diagnosis of MPFD provides opportunities for further testing and the early detection of MPFD-associated blood hypercoagulation disorders in mothers diagnosed with MPFD, and can prevent and treat serious complications that arise from blood hypercoagulation disorders.

Description

Biomarker composition for diagnosing abortion placental fibrin hyperdeposition and use thereof

The present invention relates to a biomarker composition for diagnosing placental fibrin hyperdeposition and its use.

The placenta is an organ responsible for nutrient supply, gas exchange, and waste discharge by connecting the fetus and the mother's uterine wall, and a part of the membrane surrounding the fetus is formed by adhering to the mother's endometrium. Most of the placenta that mediates the exchange of substances necessary for the survival and growth of the fetus between the fetus and the mother is tissue derived from the fetus, and the structure formed by the mother is attached to the bottom of the placenta. Specifically, a part of the fertilized egg implanted in the uterine wall grows and turns into a chorionic membrane responsible for mass transfer in the placenta, and a part of the uterus turns into a detached membrane surrounding the outermost part of the placenta (Sarah K, et al., 2015). .

As such, the placenta, which plays a key function necessary for the growth of the fetus through material exchange at the boundary between the mother and the fetus, injects blood between the placenta and the spiral artery of the mother. The villi in the placenta are immersed in the blood of the mother who has spouted. When the villi submerged in the blood are observed under a microscope, blood of the fetus flows through the blood vessels of the fetus inside the villi, and blood of the mother exists outside the villi. The transfer of nutrients, including carbon dioxide and oxygen, occurs between the mother's blood and the fetal blood existing inside and outside the villi.

On the other hand, there are cases where fibrinoids are over-deposited around the villi that separate the mother and fetal blood, which is called placental fibrin deposition (MPFD). If the placenta is full of fibrinoids, the placenta will turn yellow and the fibrinoids in the space between the villi will lose the maternal-fetal interface, preventing the exchange of gas and nutrients between the fetus and mother, thereby completely losing placenta function. Is done.

The fetus of the mother who is MPFD develops delayed in 24 to 100%, dies with a probability of 13 to 50%, and premature birth occurs in 26 to 60%. Even if the fetus survives and is given birth, severe brain damage occurs. The frequency of such MPFD is 0.005 to 0.5%, which is a rare disease, but in the case of MPFD, there are cases where the fetus dies suddenly in the second half of pregnancy after there are no specific signs during pregnancy. This may happen. In the case of surviving children, severe brain damage has occurred, and pediatricians may also face medical malpractice.

However, to date, the cause of MPFD has not been well understood, and MPFD in abortion in early pregnancy has been rarely studied worldwide.

Thus, the present inventors were studying a method for diagnosing placental fibrin hyperdeposition in the placenta of a pregnant woman, while finding specific proteins expressed only in the blood of the mother diagnosed with fibrin type MPFD (f-MPFD). The invention was completed.

An object of the present invention is to provide a biomarker composition for diagnosing placental fibrin hyperdeposition associated with abortion.

In order to achieve the above object, the present invention is the group consisting of CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, LGALS1 and IGFBP4 Provided is a biomarker composition for diagnosing lactic acid placenta fibrin hyperdeposition or habitual miscarriage, which includes any one or more proteins selected from active ingredients.

In addition, the present invention consists of CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, LGALS1 and IGFBP4 in blood collected from mothers. Measuring any one or more protein detection amounts selected from the group; And determining the mother as MPFD or addictive abortion when the protein detection amount is higher than the detection amount of the protein present in the normal mother's blood.

In addition, the present invention comprises the steps of measuring the degree of sensitization to HLA (human leukocyte antigen) in blood collected from a mother; Comparing the level of sensitization to HLA with the level of sensitization to HLA in normal maternal blood; And determining the mother as MPFD when the degree of sensitization to HLA is higher than that of normal mother blood to HLA.

In addition, the present invention comprises the steps of performing C4d (Complement component 4d) immunostaining in blood collected from mothers determined to be MPFD by performing a method for providing information necessary for the diagnosis of MPFD; And if the result of the immunostaining, C4d positive (C4d immunopositive) provides a method for providing information necessary for diagnosing addictive abortion, comprising the step of determining the mother as a habitual miscarriage (recurrent miscarriage).

In addition, the present invention is selected from the group consisting of CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, LGALS1 and IGFBP4 Provided is a kit for diagnosing MPFD or addictive miscarriage comprising an antibody or fragment thereof that binds to the above protein as an active ingredient.

The protein included as an active ingredient in the biomarker composition for diagnosing abortion placental fibrin hyperdeposition according to the present invention is specifically found only in the blood of mothers diagnosed with fibrin type MPFD (f-MPFD) and is useful for diagnosis with f-MPFD It can be utilized and can prevent addictive miscarriage according to f-MPFD in advance. In addition, through the diagnosis of MPFD, an MPFD-related blood hypercoagulant disease may be additionally examined in the mother diagnosed with MPFD, thereby providing an opportunity to discover it in advance, and prevent and treat serious complications associated with the blood coagulation disease.

1 shows the MPFD diagnostic criteria with placenta microscopic observation images.

Figure 2 is a microscopic observation of the placenta of the mother diagnosed with MPFD by H & E staining. The fibrin is shown in pink, and it can be seen that the fibrin was overdeposited around the placental villi.

Figure 3 is an automatic image analysis of the placenta of a mother diagnosed with MPFD. At this time, Bg means the background of the digital slide image during H & E staining, and v stands for "villi" and means placental villi.

Figure 4 shows the placenta of a normal mother and the mother's placenta diagnosed with MPFD, along with microscopic observation images.

Figure 5 shows the results of positive and negative reactions during microscopy of C4d immunochemical staining.

6 is a graph showing whether abortion is repeated according to the C4d immunostaining reaction of abortion group mothers.

7A and 7B show that the placenta of the mother was stained with H & E, fibrin, and C4d and observed under a microscope when diagnosing the 6th and 7th miscarriage of the mother diagnosed with the fibrin type MPFD showing a positive response as a result of C4d immunostaining.

8A and 8B are graphs confirming whether an antibody against HLA antigen was produced by sensitization to HLA antigen (human leukocyte antigen) through pregnancy through HLA panel.

Figure 9a shows the results of observing the blood of the mother diagnosed with fibrin type MPFD with H & E, anti-fibrin antibody or anti-collagen type IV antibody under a microscope (x40 and x200).

Figure 9b shows the results of observing the blood of the mother diagnosed with matrix type MPFD with H & E, anti-fibrin antibody or anti-collagen type IV antibody under a microscope (x40 and x200).

10 is a graph showing the probability of addictive miscarriage among mothers diagnosed with MPFD and increased intervillous fibrin (IFF).

11 is a graph showing the probability of repeated miscarriage according to the MPFD type.

12 is a graph showing the results of experiments on the placental chromosome abnormality for mothers diagnosed with f-MPFD, m-MPFD, and fibrin deposition-free abortion.

FIG. 13 is a diagram showing the results of protein analysis of plasma obtained from f-MPFD, miscarriage control (MC), and normal control (NC) groups.

14 is a quantitative analysis of the protein by LFQ (Label free quantification), statistically shows that the p value value is less than 0.05 and the difference in protein amount between groups by 2 times or more is shown in a volcano plot.

In one aspect, the present invention is selected from the group consisting of CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, LGALS1 and IGFBP4 Provided is a biomarker composition for diagnosing lactic acid placenta fibrin hyperdeposition or habitual miscarriage, which contains at least one protein as an active ingredient.

In this case, the CCT3 is a polypeptide having the amino acid sequence of SEQ ID NO: 1, the CCT7 is a polypeptide having the amino acid sequence of SEQ ID NO: 3, the PSMA2 is a polypeptide having the amino acid sequence of SEQ ID NO: 5, or the HSPA4 is The polypeptide having the amino acid sequence of SEQ ID NO: 7, the RAD23B is the polypeptide having the amino acid sequence of SEQ ID NO: 9, the UBE2D3 is the polypeptide having the amino acid sequence of SEQ ID NO: 11, or the HNRNPC is the amino acid of SEQ ID NO: 13 A polypeptide having a sequence, the EFTUD2 is a polypeptide having the amino acid sequence of SEQ ID NO: 15, the MBP is a polypeptide having the amino acid sequence of SEQ ID NO: 17, or the AHNAK is a polypeptide having the amino acid sequence of SEQ ID NO: 19 Or, the HSD17B10 is the amino acid sequence of SEQ ID NO: 21 Or the IL18 is a polypeptide having the amino acid sequence of SEQ ID NO: 23, the RPL22 is a polypeptide having the amino acid sequence of SEQ ID NO: 25, or the SERBP1 is a polypeptide having the amino acid sequence of SEQ ID NO: 27 , The PCOLCE is a polypeptide having the amino acid sequence of SEQ ID NO: 29, the ACAA2 is a polypeptide having the amino acid sequence of SEQ ID NO: 31, the FASN is a polypeptide having the amino acid sequence of SEQ ID NO: 33, or the LGALS1 is the sequence It may be a polypeptide having the amino acid sequence of SEQ ID NO: 35, or the IGFBP4 may be a polypeptide having the amino acid sequence of SEQ ID NO: 37.

The term "CCT3" used herein is an abbreviation of "chaperonin containing TCP1, subunit 3" and is also known as CCTG, PIG48, TRIC5, CCT-gamma or TCP-1-gamma. The CCT3 protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 1, and the CCT3 protein may have about 70%, 80%, 90%, or 95% or more homology with the amino acid sequence of SEQ ID NO: 1. . Meanwhile, the gene encoding the CCT3 protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 1, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 2. The base sequence encoding the CCT3 protein may have about 70%, 80%, 90%, or 95% or more homology with the base sequence of SEQ ID NO: 2.

The term "CCT7" used herein is an abbreviation of "chaperonin containing TCP1, subunit 7" and is also called CCTH, CCTETA, NIP7-1 or TCP1ETA. The CCT7 protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 3, and the CCT7 protein may have about 70%, 80%, 90% or 95% or more homology with the amino acid sequence of SEQ ID NO: 3. . Meanwhile, the gene encoding the CCT7 protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 3, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 4. The base sequence encoding the CCT7 protein may have about 70%, 80%, 90%, or 95% or more homology with the base sequence of SEQ ID NO: 4.

The term "PSMA2" used herein is an abbreviation of "proteasome subunit alpha type 2" and is also called MU, HC3, PSC2 or PMSA2. The PSMA2 protein of the present invention may be a polypeptide having an amino acid sequence of SEQ ID NO: 5, and the PSMA2 protein may have a homology of about 70%, 80%, 90%, or 95% or more with the amino acid sequence of SEQ ID NO: 5 . Meanwhile, the gene encoding the PSMA2 protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 5, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 6. The base sequence encoding the PSMA2 protein may have about 70%, 80%, 90%, or 95% or more homology with the base sequence of SEQ ID NO: 6.

The term "HSPA4" as used herein is an abbreviation of "heat shock protein family A member 4" and is also known as RY, APG-2, HSPH2, hsp70, hsp70RY, HEL-S-5a or HS24 / P52. The HSPA4 protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 7, and the HSPA4 protein may have at least about 70%, 80%, 90%, or 95% homology to the amino acid sequence of SEQ ID NO: 7. . Meanwhile, the gene encoding the HSPA4 protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 7, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 8. The base sequence encoding the HSPA4 protein may have about 70%, 80%, 90%, or 95% or more homology with the base sequence of SEQ ID NO: 8.

The term "RAD23B" used herein is an abbreviation of "RAD23 homolog B, nucleotide excision repair protein" and is also called P58, HR23B or HHR23B. The RAD23B protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 9, and the RAD23B protein may have about 70%, 80%, 90% or 95% or more homology with the amino acid sequence of SEQ ID NO: 9. . Meanwhile, the gene encoding the RAD23B protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 9, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 10. The base sequence encoding the RAD23B protein may have about 70%, 80%, 90%, or 95% or more homology with the base sequence of SEQ ID NO: 10.

The term "UBE2D3" used herein is an abbreviation of "ubiquitin-conjugating enzyme E2 D 3" and is also called UBC4 / 5, UBCH5C or E2 (17) KB3. The UBE2D3 protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 11, and the UBE2D3 protein may have about 70%, 80%, 90% or 95% or more homology to the amino acid sequence of SEQ ID NO: 11. . Meanwhile, the gene encoding the UBE2D3 protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 11, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 12. The base sequence encoding the UBE2D3 protein may have about 70%, 80%, 90% or 95% or more homology with the base sequence of SEQ ID NO: 12.

The term "HNRNPC" as used herein stands for "heterogeneous nuclear ribonucleoprotein C (C1 / C2)" and is also called C1, C2, HNRNP, HNRPC or SNRPC. The HNRNPC protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 13, and the HNRNPC protein may have a homology of at least 70%, 80%, 90% or 95% with the amino acid sequence of SEQ ID NO: 13 . Meanwhile, the gene encoding the HNRNPC protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 13, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 14. The base sequence encoding the HNRNPC protein may have about 70%, 80%, 90% or 95% or more homology with the base sequence of SEQ ID NO: 14.

The term "EFTUD2" used herein is an abbreviation of "elongation factor Tu GTP binding domain containing 2" and is also known as MFDM, MFDGA, Snu114, Snrp116, SNRNP116 or U5-116KD. The EFTUD2 protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 15, and the EFTUD2 protein may have about 70%, 80%, 90% or 95% or more homology with the amino acid sequence of SEQ ID NO: 15. . Meanwhile, the gene encoding the EFTUD2 protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 15, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 16. The base sequence encoding the EFTUD2 protein may have a homology of at least about 70%, 80%, 90%, or 95% with the base sequence of SEQ ID NO: 16.

The term "MBP" used herein stands for "myelin basic protein", and the MBP protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 17. In addition, the MBP protein may have about 70%, 80%, 90%, or 95% or more homology with the amino acid sequence of SEQ ID NO: 17. Meanwhile, the gene encoding the MBP protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 17, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 18. The base sequence encoding the MBP protein may have about 70%, 80%, 90% or 95% or more homology with the base sequence of SEQ ID NO: 18.

The term "AHNAK" used herein is an abbreviation of "AHNAK nucleoprotein" and is also called PM227 or AHNAKRS. The AHNAK protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 19. The AHNAK protein may have at least about 70%, 80%, 90%, or 95% homology to the amino acid sequence of SEQ ID NO: 19. Meanwhile, the gene encoding the AHNAK protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 19, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 20. The base sequence encoding the AHNAK protein may have a homology of at least about 70%, 80%, 90%, or 95% with the base sequence of SEQ ID NO: 20.

The term "HSD17B10" as used herein stands for "hydroxysteroid 17-beta dehydrogenase 10", ABAD, CAMR, ERAB, HCD2, MHBD, HADH2, MRPP2, MRX17, MRX31, SCHAD, MRXS10, SDR5C1, HSD10MD, 17b-HSD10 or Also known as DUPXp11.22. The HSD17B10 protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 21, and the HSD17B10 protein may have at least about 70%, 80%, 90%, or 95% homology to the amino acid sequence of SEQ ID NO: 21. . Meanwhile, the gene encoding the HSD17B10 protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 21, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 22. The base sequence encoding the HSD17B10 protein may have a homology of at least about 70%, 80%, 90%, or 95% with the base sequence of SEQ ID NO: 22.

As used herein, the term "IL18" stands for "interleukin 18" and is also known as IGIF, IL-18, IL-1g or IL1F4. The IL18 protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 23, and the IL18 protein may have a homology of at least about 70%, 80%, 90%, or 95% with the amino acid sequence of SEQ ID NO: 23. . Meanwhile, the gene encoding the IL18 protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 23, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 24. The nucleotide sequence encoding the IL18 protein may have about 70%, 80%, 90%, or 95% or more homology with the nucleotide sequence of SEQ ID NO: 24.

The term "RPL22" used herein is an abbreviation of "ribosomal protein L22" and is also called EAP, L22, HBP15 or HBP15 / L22. The RPL22 protein of the present invention may be a polypeptide having an amino acid sequence of SEQ ID NO: 25, and the RPL22 protein may have a homology of about 70%, 80%, 90%, or 95% or more with the amino acid sequence of SEQ ID NO: 25. . Meanwhile, the gene encoding the RPL22 protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 25, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 26. The base sequence encoding the RPL22 protein may have about 70%, 80%, 90%, or 95% or more homology with the base sequence of SEQ ID NO: 26.

The term "SERBP1" used herein is also abbreviation of "SERPINE1 mRNA binding protein 1" and is also known as CGI-55, CHD3IP, HABP4L, PAIRBP1 or PAI-RBP1. The SERBP1 protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 27, and the SERBP1 protein may have a homology of at least 70%, 80%, 90%, or 95% with the amino acid sequence of SEQ ID NO: 27. . Meanwhile, the gene encoding the SERBP1 protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 27, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 28. The base sequence encoding the SERBP1 protein may have about 70%, 80%, 90%, or 95% or more homology with the base sequence of SEQ ID NO: 28.

The term "PCOLCE" as used herein stands for "procollagen C-endopeptidase enhancer" and is also called PCPE, PCPE1 or PCPE-1. The PCOLCE protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 29, and the PCOLCE protein may have a homology of at least about 70%, 80%, 90%, or 95% with the amino acid sequence of SEQ ID NO: 29. . Meanwhile, the gene encoding the PCOLCE protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 29, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 30. The base sequence encoding the PCOLCE protein may have about 70%, 80%, 90%, or 95% or more homology with the base sequence of SEQ ID NO: 30.

The term "ACAA2" used herein is also known as DSAEC for "acetyl-CoA acyltransferase 2". ACAA2 protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 31, the ACAA2 protein may have a homology of at least about 70%, 80%, 90% or 95% with the amino acid sequence of SEQ ID NO: 31 . Meanwhile, the gene encoding the ACAA2 protein, which is the polypeptide having the amino acid sequence of SEQ ID NO: 31, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 32. The base sequence encoding the ACAA2 protein may have about 70%, 80%, 90%, or 95% or more homology with the base sequence of SEQ ID NO: 32.

The term "FASN" used herein is an abbreviation of "fatty acid synthase" and is also called FAS, OA-519 or SDR27X1. The FASN protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 33, and the FASN protein may have about 70%, 80%, 90% or 95% or more homology with the amino acid sequence of SEQ ID NO: 33. . Meanwhile, the gene encoding the FASN protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 33, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 34. The base sequence encoding the FASN protein may have at least about 70%, 80%, 90%, or 95% homology with the base sequence of SEQ ID NO: 34.

The term "LGALS1" used herein is an abbreviation of "lectin, galactoside-binding, soluble, 1", and the LGALS1 protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 35. In addition, the LGALS1 protein may have about 70%, 80%, 90%, or 95% or more homology with the amino acid sequence of SEQ ID NO: 35. Meanwhile, the gene encoding the LGALS1 protein, which is the polypeptide having the amino acid sequence of SEQ ID NO: 35, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 36. The base sequence encoding the LGALS1 protein may have about 70%, 80%, 90%, or 95% or more homology with the base sequence of SEQ ID NO: 36.

The term "IGFBP4" used herein is an abbreviation of "insulin-like growth factor binding protein 4" and is also known as BP-4, IBP4, IGFBP-4 or HT29-IGFBP. The IGFBP4 protein of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 37, and the IGFBP4 protein may have about 70%, 80%, 90% or 95% or more homology with the amino acid sequence of SEQ ID NO: 37. . Meanwhile, the gene encoding the IGFBP4 protein, which is a polypeptide having the amino acid sequence of SEQ ID NO: 37, may be a polynucleotide having the nucleotide sequence of SEQ ID NO: 38. The nucleotide sequence encoding the IGFBP4 protein may have about 70%, 80%, 90%, or 95% or more homology with the nucleotide sequence of SEQ ID NO: 38.

Specifically, the biomarker composition for diagnosing abortion placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN , LGALS1 and IGFBP4 may include any one protein selected from the group consisting of active ingredients. In one example, the MPFD diagnostic biomarker composition of the present invention may include CCT3 as an active ingredient, but is not limited thereto.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any two proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. In one example, the MPFD diagnostic biomarker composition of the present invention may include CCT3 and PCOLCE as active ingredients, but is not limited thereto.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any three proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. For example, the biomarker composition for diagnosing MPFD of the present invention may include CCT3, CCT7, and PCOLCE as active ingredients, but is not limited thereto.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any four proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. For example, the biomarker composition for diagnosing MPFD of the present invention may include CCT3, CCT7, ACAA2, and PCOLCE as active ingredients, but is not limited thereto.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any five proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. In one example, the biomarker composition for diagnosing MPFD of the present invention may include UBE2D3, HNRNPC, EFTUD2, LGALS1 and IGFBP4 as active ingredients, but is not limited thereto.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any six proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. For example, the biomarker composition for diagnosing MPFD of the present invention may include CCT3, UBE2D3, HNRNPC, EFTUD2, LGALS1 and IGFBP4 as active ingredients, but is not limited thereto.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any seven proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. For example, the biomarker composition for diagnosing MPFD of the present invention may include CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, and FASN as active ingredients, but is not limited thereto.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Eight proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. In one example, the biomarker composition for diagnosing MPFD of the present invention may include HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, and HSD17B10 as active ingredients.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any nine proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. For example, the biomarker composition for diagnosing MPFD of the present invention may include CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2 and MBP as active ingredients, but is not limited thereto.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any ten proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. In one example, the biomarker composition for diagnosing MPFD of the present invention may include CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP and IGFBP4 as active ingredients, but is not limited thereto.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any eleven proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. In one example, the biomarker composition for diagnosing MPFD of the present invention may include PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18 and RPL22 as active ingredients, but is not limited thereto.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any twelve proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. For example, the biomarker composition for diagnosing MPFD of the present invention may include PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22 and IGFBP4 as active ingredients, but is not limited thereto.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any 13 proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. As an example, the biomarker composition for diagnosing MPFD of the present invention may include UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, and LGALS1 as active ingredients, but is not limited thereto. no.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any fourteen proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. In one example, the biomarker composition for diagnosing MPFD of the present invention may include UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, LGALS1 and IGFBP4 as active ingredients, It is not limited.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any fifteen proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. For example, the biomarker composition for diagnosing MPFD of the present invention may include CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, PCOLCE, ACAA2, FASN, LGALS1 and IGFBP4 as active ingredients, It is not limited to this.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any sixteen proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. In one example, the biomarker composition for diagnosis of MPFD of the present invention may include CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, PCOLCE, HSD17B10, ACAA2, FASN, LGALS1 and IGFBP4 as active ingredients. However, it is not limited thereto.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Any seventeen proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. In one example, the biomarker composition for diagnosis of MPFD of the present invention includes CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, and LGALS1 as active ingredients. You can, but are not limited to this.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, Eighteen proteins selected from the group consisting of LGALS1 and IGFBP4 may be included as active ingredients. In one example, the biomarker composition for diagnosing MPFD of the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN and LGALS1 It may include, but is not limited to this.

In addition, the biomarker composition for diagnosing abortive placental fibrin hyperdeposition according to the present invention is CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, LGALS1 and IGFBP4 proteins may be included as active ingredients. When the biomarker composition for diagnosing MPFD of the present invention includes all 19 proteins according to the present invention as an active ingredient, it can be used as a biomarker for diagnosing MPFD more effective than a combination of other proteins.

The term "massive perivillous fibrin deposition (MPFD)" as used herein refers to a case where fibrinoids are overdeposited around the villi that separate mother and fetal blood. The MPFD is classified into a fibrin type and a matrix type. In the case of mothers diagnosed with fibrin type MPFD, the surrounding villi in the placenta are deposited with fibrin, which has a function of blood clotting. In addition, in the case of a mother diagnosed with matrix type MPFD, the surrounding villi in the placenta are deposited with an extracellular matrix such as collagen type IV and glycoproteins secreted from the placental cells, the trophoblast. In many embodiments of the present invention, the mother diagnosed with the fibrin type MPFD was named f-MPFD, and the mother diagnosed with the matrix type MPFD was named m-MPFD.

The term "biomarker" as used herein is an anatomical, physiological, biochemical or molecular parameter related to the presence of a particular physiological condition or progression. Biomarkers are detectable and measurable by a variety of methods including laboratory assays and medical imaging. When a biomarker is a marker of or indicates an abnormal progression or disease or other condition in an individual, the biomarker is generally a marker of the normal progression or disease or other condition in the individual or the expression level or value of the biomarker indicating it It is described as either over-expressed or under expressed compared to.

The term "diagnosis" as used herein determines the susceptibility of an individual to a particular disease or condition, determining whether an individual currently has a specific disease or condition, or having a specific disease or condition Determining the prognosis of an individual, or terrametrics (eg, monitoring an individual's condition to provide information about treatment efficacy).

In another aspect, the present invention, CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, LGALS1 and IGFBP4 in blood collected from mothers Measuring any one or more protein detection amounts selected from the group consisting of; And determining the mother as MPFD or addictive abortion when the protein detection amount is higher than the detection amount of the protein present in the normal mother's blood.

In this case, the protein detection amount measurement may be performed by a liquid chromatography-mass spectrometry (LC-MS) technique or an enzyme-linked immunosorbent assay (ELISA) technique.

In addition, the present invention for the diagnosis of MPFD, 1) measuring the degree of sensitization (sensitization) for HLA (human leukocyte antigen) in blood collected from mothers; 2) comparing the level of sensitization to HLA with the level of sensitization to HLA in normal maternal blood; And 3) when the sensitization level for the HLA is higher than the normal sensitization level for the HLA, the step of determining the mother as MPFD may be additionally performed.

In one embodiment of the present invention, in order to confirm whether or not the mother was sensitized to HLA antigen through pregnancy and produced an antibody against the HLA antigen, blood was paneled with HLA class I and HLA class II. To react. As a result of the experiment, it was found that the mothers diagnosed with MPFD had an increased HLA panel reactive antibody (PRA) than the mothers of other groups, and through this, the amount of HLA PRA could be the standard for MPFD diagnosis.

In addition, the present invention for the diagnosis of habitual miscarriage, 1) performing C4d (Complement component 4d) immunostaining in blood collected from mothers determined to be MPFD by the diagnostic method according to the present invention; And 2) as a result of performing the immunostaining, in the case of C4d positive (C4d immunopositive), the step of determining the mother of step 1) as a habitual miscarriage may be additionally performed.

In one embodiment of the present invention, it was hypothesized that the cause of the development of MPFD would be a rejection reaction to the fetus, and to confirm the hypothesis, immunostaining was performed using C4d, a marker related to organ transplant rejection, to abortion mothers. As a result of the experiment, C4d immunostaining showed that all mothers diagnosed with a positive fibrin-type MPFD experienced at least three repeated abortions, whereas all mothers diagnosed with a fibrin-type MPFD having a C4d negative response had repeated abortions. Did not experience. Through this, it was found that the mothers who were diagnosed with the fibrin type MPFD and showed positive results as a result of C4d immunostaining were highly likely to be repeatedly aborted.

In another aspect, the present invention is selected from the group consisting of CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, LGALS1 and IGFBP4 It provides a kit for diagnosing MPFD or habitual miscarriage comprising an antibody or fragment thereof that binds to any one or more proteins as an active ingredient.

The term "antibody" as used herein is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. Antibodies in the present invention are from the group consisting of CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, LGALS1 and IGFBP4 of the present invention. It means an antibody that specifically binds to any one or more proteins selected, and the antibody can be prepared according to a conventional method in the art. The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies are included. The antibody refers to a complete form with two full-length light chains and two full-length heavy chains. In addition, the antibody also includes special antibodies such as humanized antibodies.

In addition, the kit of the present invention, the antibody specifically binding to the marker component, a secondary antibody conjugate (conjugate) conjugated with a marker to be colored by reaction with a substrate, a substrate solution to develop a color reaction with the marker, a washing solution, and It may include an enzyme reaction stop solution, etc., and may be manufactured in a number of separate packaging or compartments containing reagent components used. Specifically, the kit may be a lateral flow kit, and more specifically, may be a rapid kit, but is not limited thereto.

The rapid kit may include a sample pad to which a liquid sample is applied, a membrane on which the antibody is immobilized, and an absorption pad capable of absorbing a liquid sample, but is not limited thereto. The liquid sample may be mother's whole blood, serum, plasma, saliva, other body fluids (urine, etc.), and specifically, may be mother's plasma, but is not limited thereto.

Hereinafter, the present invention will be described in detail by the following preparation examples and examples. However, the following preparation examples and examples are only for illustrating the present invention, and the present invention is not limited by them.

실시예 1. 유산 집단 수집Example 1. Collection of heritage groups

A miscarriage cohort was constructed by collecting blood from the placenta and mothers aborted from the Hamchoon Women's Clinic Heritage Clinic. Specifically, from March 2012 to June 2016, we collected cases that were aborted at the Chun Chun Women's Clinic and performed curettage, and 582 samples were cured through curettage from a mother who found 562 miscarriage in 4 years and 4 months. Obtained. Pathology cassettes were prepared from samples obtained by curettage, which were fixed with formalin and embedded in paraffin. In addition, the blood of the mother whose abortion was found was separated in the form of plasma and serum and stored at -80 ° C.

실시예 2. 유산 집단 분류Example 2. Classification of heritage groups

The heritage group collected in Example 1 was classified into three groups as follows. First, among the mothers diagnosed with abortion placental fibrin hyper deposition, seven women (fibrin type) and normal placental chromosomes (Hamchun Women's Clinic) were selected as the first group, and this group was named f-MPFD. Did. At this time, more than 25% of the total villi in the placenta were diagnosed with fibrin deposition as MPFD (Katzman PJ, Dev Pathol, 2002). This is shown in FIG. 1. In order to measure fibrin deposition more objectively, an automated imaging analysis technique was used. The case where fibrin is overdeposited around the villi in the placenta is shown in FIG. 2, and the automatic image analysis results are shown in FIG. 3. In addition, the placenta of a normal mother and the mother's placenta diagnosed with MPFD are shown in FIG. 4.

In addition, 18 mothers (Hamchun Women's Clinic), whose age, miscarriage, and gestational weeks were matched and whose placenta chromosomes were normal were selected as the second group as miscarriage control. This group was named MC. It is shown in Table 1 below. Lastly, two healthy mothers who were undergoing prenatal examination at Seoul Asan Hospital and did not have any particular disease were selected as the third group as a normal control, and this group was named NC.

[Table 1]

실험예 1. C4d 면역화학염색Experimental Example 1. C4d immunochemical staining

Since pregnancy is a type of organ transplant, it is hypothesized that the cause of MPFD may be rejection of the fetus. In the abortion group of Example 2, immunostaining was performed using C4d, a marker related to an organ transplant rejection response. The results of C4d positive and negative are shown in FIG. 5.

In addition, as a result of confirming the relationship between the C4d immune response and recurrent miscarriage, all mothers diagnosed with fibrin-type MPFD that showed a positive response as a result of C4d immunostaining experienced three or more repeated miscarriage. On the other hand, all mothers diagnosed with fibrin-type MPFD with a C4d negative response did not experience repeated abortion. This is shown in FIG. 6. Through this, it was found that the mothers who were diagnosed with the fibrin type MPFD and showed positive results as a result of C4d immunostaining were highly likely to be repeatedly aborted.

On the other hand, when the mother diagnosed with the fibrin type MPFD showing a positive response as a result of C4d immunostaining as described above was diagnosed with the 6th and 7th miscarriage, the mother's placenta was stained with H & E, fibrin and C4d and observed under a microscope 7A and 7B. As shown in FIGS. 7A and 7B, two mothers diagnosed with fibrin-type MPFD that showed a positive response as a result of C4d immunostaining showed the same pathology pattern in both the 6th and 7th abortions.

실험예 2. HLA 항체 스크리닝Experimental Example 2. HLA antibody screening

In order to confirm the hypothesis that the cause of the occurrence of MPFD established in Experimental Example 1 would be the rejection reaction to the fetus, the maternal blood rejected the fetus using the human leukocyte antigen (HLA), a tissue-compatible antigen used for organ transplantation. It was confirmed that there is. This experiment was carried out in the f-MPFD and MC groups among the heritage groups classified in Example 2. In addition, for this experiment, the abortion group was divided into three groups as follows: 14 women diagnosed with MPFD, 7 women with increased fibrin deposition between villi (IIF) and 7 abortion control mothers 18 people.

In order to confirm whether the mother of each group was sensitized to HLA antigens through pregnancy and produced antibodies against HLA antigens, a panel consisting of HLA class I and HLA class II blood (LABScreen Mixed class I) and II assay (Luminex Corporation, Austin, Texas); Luminex LX 200 (Luminex Corporation, Austin, Texas). At this time, in order to determine whether to produce HLA antibodies, LABScreen Mixed Class I and II analysis methods (Luminex Corporation, Austin, Texas, US) and Luminex LX 200 (Luminex Corporation, Austin, Texas, US) were used. The results are shown in FIGS. 8A and 8B.

8A and 8B, mothers diagnosed with MPFD had an increased HLA panel reactive antibody (PRA) than mothers with IIF. In addition, in the miscarriage group classified in Example 2, the HLA panel-reactive antibody was significantly increased in the mother diagnosed with f-MPFD than in the MC group. In addition, in Example 2, the mothers diagnosed with f-MPFD, that is, the 7 mothers with normal placental chromosomes diagnosed with fibrin type MPFD (Hamchun Women's Clinic) all had a positive response as a result of C4d immunostaining At the same time, it showed a positive reaction with HLA panel reactive antibody.

실험예 3. MPFD 질환과 반복 유산의 관계Experimental Example 3. Relationship between MPFD disease and repeated abortion

The incidence of MPFD and the recurrent miscarriage rate in mothers diagnosed with MPFD were confirmed through the mothers in which 562 miscarriers recruited in Example 1 were found.

Of the 562 abortions found, 15 were diagnosed with MPFD and had an incidence of MPFD of 2.7%. In addition, among 15 mothers diagnosed with MPFD, fibrin-type MPFD (f-MPFD) was diagnosed with 11 women, accounting for 73% of mothers diagnosed with MPFD. In addition, of the 15 mothers diagnosed with MPFD, 4 were maternal diagnosed with matrix type MPFD (m-MPFD), accounting for 27% of mothers diagnosed with MPFD. H & E, anti-fibrin antibodies (anti fibrin ab, Biorbyt, Cambridge, UK) or anti-collagen type IV antibodies (anti collagen type IV ab, Biorbyt, Cambridge, etc.) were used for the blood of mothers diagnosed with the fibrin type MPFD and matrix type MPFD. UK), and the results observed according to the magnification (x40 and x200) under a microscope are shown in FIGS. 9A and 9B. Through this, it was found that the anti-fibrin antibody and the anti-collagen type IV antibody are markers that can easily distinguish the type of MPFD.

On the other hand, the number of mothers with increased fibrin deposition (IIF) between villi was 7, indicating an incidence of IIF of 1.25% among mothers with abortion. Of these, fibrin type IIF was found in 4 patients, and matrix type IIF was found in 3 patients.

In addition, of the 15 women diagnosed with MPFD, previously experienced 2 miscarriages, and this time, the third miscarriage was 12, accounting for 80% of mothers diagnosed with MPFD. In addition, of the 7 women diagnosed with IIF, they experienced 2 miscarriages before, and this time, the third miscarriage was 2 women, accounting for 28.6% of the mothers diagnosed with IIF. This is shown in Figure 10.

The probability that a miscarriage is repeated more than once by chance is less than 1%, and that a miscarriage is repeated three times indicates that the miscarriage is continuing and will continue to repeat. In the medical field, this is called habitual abortion, and through this experiment, it was found that 80% of mothers diagnosed with MPFD are addictive abortion.

실험예 3.1. 타입별 MPFD 질환과 반복 유산의 관계Experimental Example 3.1. Relationship between MPFD disease and recurring miscarriage by type

Repeated abortion rates according to MPFD types were confirmed in mothers diagnosed with MPFD. Of 11 women diagnosed with fibrin-type MPFD (f-MPFD), 6 had more than 4 miscarriages. In other words, the incidence of habitual miscarriage was 54.5% in mothers diagnosed with f-MPFD. On the other hand, of the 4 women diagnosed with matrix type MPFD (m-MPFD), none of them had more than 4 miscarriages. This is shown in FIG. 11. Through this, it was found that mothers diagnosed with f-MPFD are more likely to have repeated miscarriage.

실험예 3.2. MPFD 와 태반 염색체 이상 여부의 관계Experimental Example 3.2. Relationship between MPFD and placental chromosomal abnormality

In Experimental Example 3, f-MPFD, m-MPFD, and fibrin deposition-free miscarriage were tested for placental chromosomal abnormalities in mothers diagnosed with miscarriage and are shown in FIG. 12. As shown in FIG. 12, placental chromosomes in 7 out of 10 women diagnosed with f-MPFD were normal, ie, mothers diagnosed with f-MPFD had a placental chromosome normal with a probability of 70%. In addition, placental chromosomes were normal in 2 out of 3 mothers diagnosed with m-MPFD. In other words. Mothers diagnosed with m-MPFD had a 66.7% chance of normal placental chromosomes. In general, considering that more than 50% of cases occur due to a placental chromosomal abnormality, it was found that the abortion according to MPFD has a completely different mechanism of disease than normal abortion.

실험예 4. MPFD 질환과 혈액 과응고 질환의 관계Experimental Example 4. Relationship between MPFD disease and blood hypercoagulation disease

The history of mothers diagnosed with the MPFD was examined. It is shown in Table 2 below.

[Table 2]

As shown in Table 2, blood hypercoagulation diseases were found in 36% of mothers diagnosed with MPFD.

Through the above results, it can be seen that if a mother can diagnose MPFD at the time of miscarriage, a blood hypercoagulation disease test is performed on the mother diagnosed with MPFD, thereby providing an opportunity to detect the disease in advance. If blood hypercoagulation disease is present, serious complications such as pulmonary embolism and deep vein thrombosis may occur, so additional examination of blood hypercoagulation disease through MPFD diagnosis to prevent and treat it is more beneficial to maternal health It is important.

실험예 5. MPFD 진단을 위한 바이오마커 선정Experimental Example 5. Selection of biomarkers for MPFD diagnosis

실험예 5.1. f-MPFD에 특이적인 단백질 선별Experimental Example 5.1. Protein selection specific for f-MPFD

The blood of the f-MPFD (7 people), MC (18 people), and NC (2 people) groups classified in Example 2 was pooled, and 7 plasmas of the f-MPFD group and 18 plasmas of the MC group were pooled. And 2 plasmas of the NC group were prepared. Subsequently, in order to cut the protein in the prepared plasma, a process of denaturing by treating urea and DTT, that is, reduction and alkylation reactions was performed. Subsequently, a process of slicing proteins using trypsin, that is, a filter-aided sample preparation (FASP) process was performed. The urea and DTT previously used in the denaturation process were then washed using C18 resin (Thermo Scientific). Subsequently, liquid chromatography-mass spectrometry (LC-MS) was performed using Q Exactive Plus Nano LC (Thermo Fisher Scientific), and proteins were qualitatively analyzed using Proteome Discoverer 2.2 (Thermo Fisher Scientific). Protein was quantitatively analyzed by label free quantification (LFQ).

As a result of protein analysis, Venn diagrams for proteins found in the three groups are shown in FIG. 13. As shown in FIG. 13, a total of 771 proteins were found in the f-MPFD group, and 156 proteins specific to MPFD were found. In addition, among the LFQ results, statistically, a p value value of 0.05 or less and a difference in protein amount between groups by 2 times or more were indicated by a volcano plot. There were 268 proteins indicated in the volcanic chart.

As a result of the experiment, among the proteins simultaneously expressed in the MC and f-MPFD groups, 24 proteins increased more than 2 times in the MPFD group, and more than 2 times increased in the MPFD groups among proteins expressed simultaneously in the NC and f-MPFD groups. Among the proteins expressed simultaneously in the NC, MC, and f-MPFD groups, 68 proteins were found to be more than doubled in the MPFD group.

In addition, in the f-MPFD group, compared to the NC group, 237 proteins were significantly expressed more than twice, and 69 proteins were expressed less. In addition, 216 proteins were significantly expressed more than twice in the f-MPFD group compared to the MC group, and 72 proteins were expressed less. This is shown in FIG. 14. Through the above results, it was found that the proteins expressed significantly more than 2 times in the f-MPFD group can be used as a biomarker for diagnosing MPFD.

실험예 5.2. f-MPFD 진단을 위한 바이오마커 선정Experimental Example 5.2. Selection of biomarkers for f-MPFD diagnosis

As shown in Experimental Example 5.1, of the 771 proteins expressed in the f-MPFD group, 156 proteins were specifically expressed only in the f-MPFD group. In order to confirm that the 156 proteins were found in plasma, human plasma atlas of the plasma proteome database was used. After mapping the 156 proteins using the human plasma atlas, 45 mapped proteins were selected.

Thereafter, among the 45 proteins selected above, 19 proteins having a function related to the pathology of f-MPFD found through the Uniprot database with a large amount found in the f-MPFD group were selected. Table 3 shows information on the final selected biomarker candidate groups.

[Table 3]

In Table 3, CCT3, CCT7, PSMA2, HSPA4, RAD23B and UBE2D3 are molecular chaperones, heat shock proteins, ubiquitin and proteasomes associated with protein misfolding of proteins. (proteasome). In addition, HNRNPC and EFTUD2 are RNA splicing related proteins. MBP, AHNAK and HSD17B10 are proteins related to neural development.

Claims

One or more proteins selected from the group consisting of CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, LGALS1 and IGFBP4 Biomarker composition for diagnosing abortive placental fibrin deposition (MPFD) or addictive abortion.
According to claim 1,

The CCT3 is a polypeptide having the amino acid sequence of SEQ ID NO: 1, the CCT7 is a polypeptide having the amino acid sequence of SEQ ID NO: 3, the PSMA2 is a polypeptide having the amino acid sequence of SEQ ID NO: 5, or the HSPA4 is SEQ ID NO: The polypeptide having the amino acid sequence of 7, the RAD23B is the polypeptide having the amino acid sequence of SEQ ID NO: 9, the UBE2D3 is the polypeptide having the amino acid sequence of SEQ ID NO: 11, or the HNRNPC is the amino acid sequence of SEQ ID NO: 13 A polypeptide having the amino acid sequence of SEQ ID NO: 15, the MBP is a polypeptide having the amino acid sequence of SEQ ID NO: 17, or the AHNAK is a polypeptide having the amino acid sequence of SEQ ID NO: 19, The HSD17B10 has the amino acid sequence of SEQ ID NO: 21 Is a polypeptide, the IL18 is a polypeptide having the amino acid sequence of SEQ ID NO: 23, the RPL22 is a polypeptide having the amino acid sequence of SEQ ID NO: 25, or the SERBP1 is a polypeptide having the amino acid sequence of SEQ ID NO: 27, or PCOLCE is a polypeptide having the amino acid sequence of SEQ ID NO: 29, ACAA2 is a polypeptide having the amino acid sequence of SEQ ID NO: 31, the FASN is a polypeptide having the amino acid sequence of SEQ ID NO: 33, or the LGALS1 is SEQ ID NO: 35 The polypeptide having the amino acid sequence of, or the IGFBP4 is a polypeptide having the amino acid sequence of SEQ ID NO: 37, biomarker composition.
Any selected from the group consisting of CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, LGALS1 and IGFBP4 in blood collected from mothers. Measuring one or more protein detection amounts; And

A method for providing information necessary for diagnosing MPFD or addictive abortion, comprising determining that the mother is MPFD or addictive abortion when the detected amount of protein is higher than the detected amount of protein present in normal maternal blood.
According to claim 3,

The protein detection amount measurement is performed by a liquid chromatography-mass spectrometry (LC-MS) technique or an enzyme-linked immunosorbent assay (ELISA) technique, and provides information necessary for diagnosing MPFD or addictive abortion.
Measuring a degree of sensitization to HLA (human leukocyte antigen) in blood collected from a mother;

Comparing the level of sensitization to HLA with the level of sensitization to HLA in normal maternal blood; And

And determining the mother as MPFD when the degree of sensitization to HLA is higher than the level of sensitization to HLA in normal mother blood.
Complement component 4d (C4d) immunostaining in blood collected from mothers determined to be MPFD by performing the method of claim 5; And

As a result of performing the immunostaining, when C4d is positive (C4d immunopositive), comprising the step of determining the mother as a habitual miscarriage (recurrent miscarriage).
Binds to any one or more proteins selected from the group consisting of CCT3, CCT7, PSMA2, HSPA4, RAD23B, UBE2D3, HNRNPC, EFTUD2, MBP, AHNAK, HSD17B10, IL18, RPL22, SERBP1, PCOLCE, ACAA2, FASN, LGALS1 and IGFBP4 A kit for diagnosing MPFD or addictive abortion, comprising an antibody or fragment thereof as an active ingredient.
The method of claim 7,

The kit is a rapid kit, MPFD or kit for diagnosing habitual miscarriage.