KR20200058832A - Association of miR―27a A>G, miR―423 C>A, miR―449b A>G and miR―604 A>G single nucleotide polymorphism with the risk of recurrent implantation failure in a Korean women - Google Patents

Association of miR―27a A>G, miR―423 C>A, miR―449b A>G and miR―604 A>G single nucleotide polymorphism with the risk of recurrent implantation failure in a Korean women Download PDF

Info

Publication number
KR20200058832A
KR20200058832A KR1020180143428A KR20180143428A KR20200058832A KR 20200058832 A KR20200058832 A KR 20200058832A KR 1020180143428 A KR1020180143428 A KR 1020180143428A KR 20180143428 A KR20180143428 A KR 20180143428A KR 20200058832 A KR20200058832 A KR 20200058832A
Authority
KR
South Korea
Prior art keywords
mir
risk
restriction enzyme
rif
seq
Prior art date
Application number
KR1020180143428A
Other languages
Korean (ko)
Other versions
KR102143770B1 (en
Inventor
김남근
이우식
이현아
사공정현
김정오
Original Assignee
차의과학대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 차의과학대학교 산학협력단 filed Critical 차의과학대학교 산학협력단
Priority to KR1020180143428A priority Critical patent/KR102143770B1/en
Publication of KR20200058832A publication Critical patent/KR20200058832A/en
Application granted granted Critical
Publication of KR102143770B1 publication Critical patent/KR102143770B1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177Receptors; Cell surface antigens; Cell surface determinants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6827Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/545Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/158Expression markers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/178Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Hematology (AREA)
  • Epidemiology (AREA)
  • Oncology (AREA)
  • Food Science & Technology (AREA)
  • Hospice & Palliative Care (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention relates to a correlation between miR-27a A>G (rs895819), miR-423 C > A (rs6505162), miR-449b A > G (rs10061133) and miR-604 A > G (rs2368393) single nucleotide polymorphisms and a risk of recurrent implantation failures among Korean women and, more particularly, to a method and kit for distinguishing a single nucleotide polymorphism selected from miR-27a A > G, miR-449b A > G and miR-604 A > G from a subject′s DNA sample or further distinguishing a single nucleotide polymorphism of miR-423 C > A to provide information required for predicting a risk of developing recurrent implantation failures among Korean women. According to the present invention, it is possible to provide information very useful in predicting a risk of developing recurrent implantation failures among Korean women by distinguishing a single nucleotide polymorphism selected from miR-27a A > G, miR-449b A > G and miR-604 A > G from a subject′s DNA sample or further distinguishing a single nucleotide polymorphism of miR-423 C > A to provide information required for predicting a risk of developing recurrent implantation failures among Korean women. In particular, in case of using the kit of the present invention using the single nucleotide polymorphism, it is possible to more easily provide the above information.

Description

miR―27a A>G, miR―423 C>A, miR―449b A>G 및 miR―604 A>G 단일염기다형성과 한국 여성의 반복착상실패 발병 위험의 연관성{Association of miR―27a A>G, miR―423 C>A, miR―449b A>G and miR―604 A>G single nucleotide polymorphism with the risk of recurrent implantation failure in a Korean women}miR-27a A> G, miR-423 C> A, miR-449b A> G and miR-604 A> G Association between monobasic polymorphism and risk of repeated implantation failure in Korean women {Association of miR-27a A> G , miR-423 C> A, miR-449b A> G and miR-604 A> G single nucleotide polymorphism with the risk of recurrent implantation failure in a Korean women}

본 발명은 miR-27a A>G(rs895819), miR-423 C>A(rs6505162), miR-449b A>G(rs10061133) 및 miR-604 A>G(rs2368393) 단일염기다형성과 한국 여성의 반복착상실패 발병 위험의 연관성에 관한 것으로, 구체적으로 한국 여성의 반복착상실패 발병 위험 예측에 필요한 정보를 제공하기 위하여 피검자의 DNA 시료로부터 miR-27a A>G, miR-449b A>G 및 miR-604 A>G 중에서 선택된 단일염기다형성을 판별하거나, miR-423 C>A 단일염기다형성을 더 판별하는 것을 특징으로 하는 방법 및 이를 위한 키트에 관한 것이다.The present invention miR-27a A> G (rs895819), miR-423 C> A (rs6505162), miR-449b A> G (rs10061133) and miR-604 A> G (rs2368393) monobasic polymorphism and repetition of Korean women Concerning the association of the risk of implantation failure, specifically miR-27a A> G, miR-449b A> G and miR-604 from DNA samples of subjects to provide information necessary to predict the risk of repeated implantation failure in Korean women It relates to a method and a kit for determining a single base polymorphism selected from A> G or further discriminating miR-423 C> A single base polymorphism.

반복착상실패(Repeated Implantation Failure; RIF)는 체외수정시술 후에도 배아가 자궁 내 착상에 실패한 경우가 반복되는 것을 의미한다. 반복착상실패의 원인은 산모측 요인(해부학적, 자궁내막, 혈전성향증)과 배아측 요인(유전적 요인, 자궁 내 발달장애) 등으로 나눌 수 있으며, 체외수정시술에 따른 분만 성공률은 30~60% 미만인 실정이다. 우리나라 불임질환 진료인원은 2006년 14만8천명에서 2010년 18만4천명으로 5년간 약 3만6천명이 증가(연평균 5.8% 증가)하였고, 총진료비는 2006년 143억원에서 2010년 203억원으로 5년간 약 60억원이 증가(연평균 9.4% 증가)하였다. 또한 불임으로 인한 사회경제적 부담에 대해 불임부부들은 임신 및 출산을 하기 위해 대다수가 현재 4~8년을 소요한 것으로 추정되며(불임진단 받기까지 2~4년과 불임진단 후 경과기간 2~4년), 정부의 체외수정 등 보조생식 시술에 따른 의료비 지원횟수인 3회를 초과하여 4회 이상 시술을 받은 여성은 약 3명 중 1명이어서 시술비용 부담이 큼에도 불구하고(1회당 평균 445만원) 불임부부의 86.4%가 앞으로 출산할 때까지 계속해서 불임치료를 받겠다는 의향을 밝혀 보조생식시술을 포함한 불임치료에 따른 비용부담이 가정경제에 미치는 영향이 심각하다(실태조사에 응답한 불임여성의 97.8%가 비용부담이 크다고 응답).Repeated Implantation Failure (RIF) means that the embryo fails to implant in the womb even after an in vitro fertilization procedure. The causes of repeated implantation failure can be divided into maternal factors (anatomical, endometrial, thrombotic) and embryonic factors (genetic factors, developmental disorders in the uterus), etc., and the success rate of delivery according to in vitro fertilization is 30 ~ It is less than 60%. The number of medical treatments for infertility diseases in Korea increased from 148,000 in 2006 to 184,000 in 2010, an increase of 36,000 over the past five years (an average annual increase of 5.8%), and the total medical expenses from 13.4 billion in 2006 to 23.2 billion in 2010. About 5 billion won increased over the past 5 years (an annual increase of 9.4%). In addition, for the socio-economic burden of infertility, it is estimated that the majority of infertile couples currently take 4 to 8 years to get pregnant and give birth (2 to 4 years before receiving infertility diagnosis and 2 to 4 years after infertility diagnosis) ), In spite of the high burden of treatment costs (average of 4.45 million won per session), one out of three women received more than four treatments in excess of three times, which is the number of medical expenses supported by the government's in vitro fertilization procedure ) 86.4% of infertile couples indicated their intention to continue to receive infertility treatment until childbirth in the future, and the cost burden of infertility treatment, including assisted reproductive procedures, has a significant impact on the home economy (infertility women who responded to the survey) 97.8% of the respondents said that the cost is high).

한편, 현재 반복착상실패의 위험 인자로 부모의 염색체 이상, 산모의 혈전증, 호르몬이상, 감염질환, 자가면역질환, 자궁 및 나팔관 기형, 염색체 이상, 다낭성난소증후군, 조기난소부전증이 제시되고 있으나 이러한 것들은 위험 인자이지 정확한 반복착상실패의 병인으로서 제시되지 못하고 있으며, 원인불명의 반복착상실패의 위험도 및 발병률 등의 스크리닝 정보 또한 제시할 수 없는 경우가 대부분이다. 때문에 정확한 위험도 제시 및 스크리닝 검사는 아직까지 존재하지 않는 실정이다.On the other hand, chromosomal abnormalities of parents, thrombosis of mothers, hormonal abnormalities, infectious diseases, autoimmune diseases, uterine and fallopian tube malformations, chromosomal abnormalities, polycystic ovary syndrome, and early ovarian dysfunction have been suggested as risk factors for repeated implantation failure. Although it is a risk factor, it has not been suggested as an etiology of repeated repetitive failure, and in most cases, screening information, such as the risk and incidence of repeated failure, is unknown. For this reason, accurate risk presentation and screening tests have not yet existed.

Reprod Biomed Online. 2014 Jan;28(1):14-38.Reprod Biomed Online. 2014 Jan; 28 (1): 14-38.

따라서 본 발명의 주된 목적은 한국 여성의 반복착상실패 발병 위험 예측에 유용한 새로운 마커 및 이를 이용하는 방법을 제공하는데 있다.Therefore, the main object of the present invention is to provide a new marker useful for predicting the risk of developing repeated failure of Korean women and a method of using the same.

본 발명의 다른 목적은 상기 마커를 이용하는 한국 여성의 반복착상실패 발병 위험 예측에 유용한 키트를 제공하는데 있다.Another object of the present invention is to provide a kit useful for predicting the risk of recurrence of repetitive implantation failure in Korean women using the marker.

본 발명의 한 양태에 따르면, 본 발명은 한국 여성의 반복착상실패 발병 위험 예측에 필요한 정보를 제공하기 위하여 피검자의 DNA 시료로부터 miR-27a A>G(rs895819), miR-449b A>G(rs10061133) 및 miR-604 A>G(rs2368393) 중에서 선택된 단일염기다형성을 판별하는 방법을 제공한다.According to one aspect of the present invention, the present invention miR-27a A> G (rs895819), miR-449b A> G (rs10061133) from a subject's DNA sample to provide the information necessary to predict the risk of repeated implantation failure in Korean women ) And miR-604 A> G (rs2368393).

본 발명의 방법에 있어서, miR-423 C>A(rs6505162) 단일염기다형성을 더 판별하는 것이 바람직하다.In the method of the present invention, it is preferable to further discriminate miR-423 C> A (rs6505162) monobasic polymorphism.

본 발명의 다른 양태에 따르면, 본 발명은 miR-27a A>G(rs895819), miR-449b A>G(rs10061133) 및 miR-604 A>G(rs2368393) 중에서 선택된 단일염기다형성을 검출하는 수단을 포함하는 한국 여성의 반복착상실패 발병 위험 예측용 키트를 제공한다.According to another aspect of the present invention, the present invention provides a means for detecting a single base polymorphism selected from miR-27a A> G (rs895819), miR-449b A> G (rs10061133) and miR-604 A> G (rs2368393). It provides a kit for predicting the risk of repeated implantation failure in Korean women.

본 발명의 키트에 있어서, miR-423 C>A(rs6505162) 단일염기다형성을 검출하는 수단을 더 포함하는 것이 바람직하다.In the kit of the present invention, it is preferable to further include means for detecting miR-423 C> A (rs6505162) monobasic polymorphism.

본 발명의 키트에 있어서, 상기 miR-27a A>G 단일염기다형성을 검출하는 수단은 서열번호 5의 염기서열로 표시되는 올리고뉴클레오티드 프라이머, 서열번호 6의 염기서열로 표시되는 올리고뉴클레오티드 프라이머 및 DraIII 제한효소를 포함하여 이루어지는 프라이머-제한효소 세트이고, 상기 miR-449b A>G 단일염기다형성을 검출하는 수단은 서열번호 9의 염기서열로 표시되는 올리고뉴클레오티드 프라이머, 서열번호 10의 염기서열로 표시되는 올리고뉴클레오티드 프라이머 및 BsmAI 제한효소를 포함하여 이루어지는 프라이머-제한효소 세트이고, 상기 miR-604 A>G 단일염기다형성을 검출하는 수단은 서열번호 11의 염기서열로 표시되는 올리고뉴클레오티드 프라이머, 서열번호 12의 염기서열로 표시되는 올리고뉴클레오티드 프라이머 및 BssSI 제한효소를 포함하여 이루어지는 프라이머-제한효소 세트인 것이 바람직하다.In the kit of the present invention, the means for detecting the miR-27a A> G single base polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 5, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 6 and Dra III It is a primer-restriction enzyme set comprising a restriction enzyme, and the means for detecting the miR-449b A> G single base polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 9, represented by the nucleotide sequence of SEQ ID NO: 10 It is a primer-restriction enzyme set comprising an oligonucleotide primer and a Bsm AI restriction enzyme, and the means for detecting the miR-604 A> G single base polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 11, SEQ ID NO: 12 It is preferred that it is a primer-restriction enzyme set comprising an oligonucleotide primer represented by the base sequence of and a Bss SI restriction enzyme.

본 발명의 키트에 있어서, 상기 miR-423 C>A 단일염기다형성을 검출하는 수단은 서열번호 7의 염기서열로 표시되는 올리고뉴클레오티드 프라이머, 서열번호 8의 염기서열로 표시되는 올리고뉴클레오티드 프라이머 및 AciI 제한효소를 포함하여 이루어지는 프라이머-제한효소 세트인 것이 바람직하다.In the kit of the present invention, the means for detecting the miR-423 C> A single base polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 7, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 8 and Aci I It is preferably a primer-restriction enzyme set comprising a restriction enzyme.

본 발명에 따르면 miR-27a A>G, miR-449b A>G 및 miR-604 A>G 중에서 선택된 단일염기다형성을 판별하거나, miR-423 C>A 단일염기다형성을 더 판별함으로써 한국 여성의 반복착상실패 발병 위험을 예측하는데 매우 유용한 정보를 제공할 수 있다. 특히, 상기 단일염기다형성을 이용하는 본 발명의 키트를 사용할 경우 보다 용이하게 상기와 같은 정보를 제공할 수 있다.According to the present invention, the repetition of Korean women by discriminating single base polymorphism selected from miR-27a A> G, miR-449b A> G and miR-604 A> G or further discriminating miR-423 C> A single base polymorphism It can provide very useful information to predict the risk of implantation failure. In particular, when using the kit of the present invention using the single-basic polymorphism, it is possible to provide the above information more easily.

본 발명에서 이용하는 miR-27a A>G, miR-423 C>A, miR-449b A>G 및 miR-604 A>G 단일염기다형성(이하, 'SNP'로 약기)은 인간의 microRNA 유전자에서 나타날 수 있는 SNP로 미국 국립생물정보센터의 단일염기다형성 데이터베이스(NCBI dbSNP, http://http://www.ncbi.nlm.nih.gov/snp/)에 각각 rs895819, rs6505162, rs10061133, rs2368393으로 등록되어 있다.The miR-27a A> G, miR-423 C> A, miR-449b A> G and miR-604 A> G single base polymorphisms (hereinafter abbreviated as 'SNP') used in the present invention may appear in the human microRNA gene. SNPs can be registered in the National Biological Information Center's single base polymorphism database (NCBI dbSNP, http://www.ncbi.nlm.nih.gov/snp/) as rs895819, rs6505162, rs10061133, and rs2368393, respectively. It is.

miR-27a A>G SNP는 서열번호 1로 표시되는 인간의 DNA 부위에서 181번째 염기로 구분할 수 있는데, 이 염기가 아데닌(adenine) 또는 구아닌(guanine)일 수 있다.miR-27a A> G SNP can be classified as the 181th base in the human DNA region represented by SEQ ID NO: 1, which may be adenine or guanine.

miR-423 C>A SNP는 서열번호 2로 표시되는 인간의 DNA 부위에서 141번째 염기로 구분할 수 있는데, 이 염기가 시토신(cytosine) 또는 아데닌일 수 있다.miR-423 C> A SNP can be identified as the 141st base in the human DNA region represented by SEQ ID NO: 2, which may be cytosine or adenine.

miR-449b A>G SNP는 서열번호 3으로 표시되는 인간의 DNA 부위에서 141번째 염기로 구분할 수 있는데, 이 염기가 아데닌 또는 구아닌일 수 있다.miR-449b A> G SNP can be identified as the 141st base in the human DNA region represented by SEQ ID NO: 3, which may be adenine or guanine.

miR-604 A>G SNP는 서열번호 4로 표시되는 인간의 DNA 부위에서 181번째 염기로 구분할 수 있는데, 이 염기가 아데닌 또는 구아닌일 수 있다.miR-604 A> G SNP can be identified as the 181 th base in the human DNA region represented by SEQ ID NO: 4, which may be adenine or guanine.

상기와 같은 SNP의 판별은 중합효소연쇄반응-제한효소단편길이다형성(polymerase chain reaction- restriction fragment length polymorphism, PCR-RFLP) 방법, 염기서열 분석방법 등을 수행하여 이루어질 수 있다.The SNP can be determined by performing a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, a sequencing method, and the like.

중합효소연쇄반응-제한효소단편길이다형성 방법은 중합효소연쇄반응을 수행하여 대상 유전자 또는 DNA 부위를 증폭하고, 특정 제한효소로 처리하였을 때 제한효소가 인식하여 소화가 이루어지는지 또는 제한효소가 인식하지 못하여 소화가 이루어지지 않는지를 구분함으로써 염기의 다형성, 결실, 부가 또는 치환을 구분하는 방법이다. 이때 DNA의 증폭여부 및 제한효소에 의한 소화 여부는 아가로스겔을 사용한 전기영동을 통해 확인할 수 있다.The polymerase chain reaction-restriction enzyme fragment length formation method performs a polymerase chain reaction to amplify a target gene or DNA region, and when treated with a specific restriction enzyme, the restriction enzyme recognizes whether digestion is achieved or the restriction enzyme does not recognize It is a method of distinguishing polymorphism, deletion, addition or substitution of bases by distinguishing whether or not digestion is not possible. At this time, whether or not DNA is amplified and digested by restriction enzymes can be confirmed through electrophoresis using agarose gel.

염기서열 분석방법으로는 공지의 맥삼-길버트(Maxam-Gilbert) 또는 생어(Sanger)의 방법을 이용할 수 있다.As the sequencing method, a known Maxam-Gilbert or Sanger method can be used.

상기 PCR-RFLP 방법으로 다형성을 판별하는 경우에는 각 SNP 부위를 포함하는 연속된 50 내지 10,000bp 부위를 증폭하기 위한 프라이머 및 각 SNP 부위가 특정 염기인 경우의 DNA를 절단할 수 있는 제한효소를 이용할 수 있다.When polymorphism is determined by the PCR-RFLP method, primers for amplifying consecutive 50 to 10,000 bp sites including each SNP site and a restriction enzyme capable of cleaving DNA when each SNP site is a specific base are used. Can be.

보다 바람직하게는 다음의 증폭용 프라이머 및 제한효소를 사용할 수 있다.More preferably, the following primers for amplification and restriction enzymes can be used.

miR-27a A>G SNP 판별 : 서열번호 5의 정방향 프라이머 및 서열번호 6의 역방향 프라이머, DraIII 제한효소miR-27a A> G SNP discrimination: forward primer of SEQ ID NO: 5 and reverse primer of SEQ ID NO: 6, Dra III restriction enzyme

miR-423 C>A SNP 판별 : 서열번호 7의 정방향 프라이머 및 서열번호 8의 역방향 프라이머, AciI 제한효소miR-423 C> A SNP discrimination: forward primer of SEQ ID NO: 7 and reverse primer of SEQ ID NO: 8, Aci I restriction enzyme

miR-449b A>G SNP 판별 : 서열번호 9의 정방향 프라이머 및 서열번호 10의 역방향 프라이머, BsmAI 제한효소miR-449b A> G SNP discrimination: forward primer of SEQ ID NO: 9 and reverse primer of SEQ ID NO: 10, Bsm AI restriction enzyme

miR-604 A>G SNP 판별 : 서열번호 11의 정방향 프라이머 및 서열번호 12의 역방향 프라이머, BssSI 제한효소miR-604 A> G SNP discrimination: forward primer of SEQ ID NO: 11 and reverse primer of SEQ ID NO: 12, Bss SI restriction enzyme

상기 miR-27a A>G SNP 판별 프라이머 및 제한효소를 사용할 경우, 중합효소연쇄반응을 통해 182bp의 DNA 단편이 증폭되며, 제한효소를 처리하면 AA 유전자형인 경우 155bp 및 27bp의 DNA 단편, AG 유전자형인 경우 182bp, 155bp 및 27bp의 DNA 단편, GG 유전자형인 경우 182bp의 DNA 단편이 생성된다.When the miR-27a A> G SNP discriminant primer and restriction enzyme are used, a DNA fragment of 182 bp is amplified through a polymerase chain reaction, and when restriction enzyme is processed, DNA fragments of 155 bp and 27 bp in the case of AA genotype, AG genotype DNA fragments of 182 bp, 155 bp and 27 bp in case, and 182 bp DNA fragments in case of GG genotype are generated.

상기 miR-423 C>A SNP 판별 프라이머 및 제한효소를 사용할 경우, 중합효소연쇄반응을 통해 144bp의 DNA 단편이 증폭되며, 제한효소를 처리하면 CC 유전자형인 경우 122bp 및 22bp의 DNA 단편, CA 유전자형인 경우 144bp, 122bp 및 22bp의 DNA 단편, AA 유전자형인 경우 144bp의 DNA 단편이 생성된다.When the miR-423 C> A SNP discriminant primer and restriction enzyme are used, a DNA fragment of 144 bp is amplified through a polymerase chain reaction, and when processing the restriction enzyme, a DNA fragment of 122 bp and 22 bp in the CC genotype and a CA genotype In this case, DNA fragments of 144 bp, 122 bp, and 22 bp, and in the case of AA genotype, 144 bp of DNA fragments are generated.

상기 miR-449b A>G SNP 판별 프라이머 및 제한효소를 사용할 경우, 중합효소연쇄반응을 통해 119bp의 DNA 단편이 증폭되며, 제한효소를 처리하면 AA 유전자형인 경우 119bp의 DNA 단편, AG 유전자형인 경우 119bp, 97bp 및 22bp의 DNA 단편, GG 유전자형인 경우 97bp 및 22bp의 DNA 단편이 생성된다.When the miR-449b A> G SNP discriminant primer and restriction enzyme are used, a DNA fragment of 119bp is amplified through a polymerase chain reaction, and when the restriction enzyme is processed, a DNA fragment of 119bp for the AA genotype or 119bp for the AG genotype , 97 bp and 22 bp DNA fragments, and in the case of the GG genotype, 97 bp and 22 bp DNA fragments are generated.

상기 miR-604 A>G SNP 판별 프라이머 및 제한효소를 사용할 경우, 중합효소연쇄반응을 통해 243bp의 DNA 단편이 증폭되며, 제한효소를 처리하면 AA 유전자형인 경우 169bp 및 74bp의 DNA 단편, AG 유전자형인 경우 243bp, 169bp 및 74bp의 DNA 단편, GG 유전자형인 경우 243bp의 DNA 단편이 생성된다.When the miR-604 A> G SNP discriminant primer and restriction enzyme are used, a DNA fragment of 243 bp is amplified through a polymerase chain reaction, and when processing the restriction enzyme, DNA fragments of 169 bp and 74 bp in the case of AA genotype, AG genotype In this case, DNA fragments of 243 bp, 169 bp, and 74 bp, and in the case of the GG genotype, DNA fragments of 243 bp are generated.

본 발명에 의하면 상기와 같은 방법으로 miR-27a A>G, miR-423 C>A, miR-449b A>G 및 miR-604 A>G의 각 SNP를 판별하고 이를 바탕으로 한국 여성의 반복착상실패 발병 위험을 예측하기 위한 정보를 제공할 수 있다. 이는 반복착상실패 환자 및 대조군 집단의 비교 분석을 통해 확인된 유의적인 차이(p<0.05)에 기인한 것이다.According to the present invention, each SNP of miR-27a A> G, miR-423 C> A, miR-449b A> G and miR-604 A> G is determined in the same manner as described above, and based on this, repeated implantation of Korean women It can provide information to predict the risk of failure. This is attributable to a significant difference ( p <0.05) confirmed through comparative analysis of the repeat failure and control groups.

기존 학계에서는 체외수정시술 후에도 배아가 자궁 내 착상에 실패한 경우가 적어도 3회 이상 발생한 경우를 반복착상실패(이하, 'RIF'로 약기함)로 정의하고 있지만, 최근에는 이러한 착상실패가 2회 발생하더라도 연속적으로 발생할 경우 또한 RIF로 보아야 한다는 의견이 있다. 이에 본 발명에서는 착상실패가 2회 연속적으로 발생하거나 혹은 총 착상실패 횟수가 3회 이상인 경우를 RIF로 정의한다.Existing academia defines recurring implantation failure (hereinafter abbreviated as 'RIF') when embryos fail to implant in the womb even after in vitro fertilization, but these implantation failures have occurred twice recently. There is an opinion that even if it occurs continuously, it should also be viewed as RIF. Accordingly, in the present invention, a case in which the occurrence of an implantation failure occurs twice consecutively or the total number of implantation failures is 3 or more is defined as RIF.

본 발명에 따르면, 상기 각 SNP 판별 결과를 바탕으로 다음과 같이 한국 여성의 RIF 발병 위험을 예측할 수 있다.According to the present invention, it is possible to predict the risk of developing RIF in Korean women as follows based on the results of each SNP discrimination.

1) 피검자의 miR-27a A>G 유전자형이 AG 유전자형인 경우 AA 유전자형(우세 유전자형)인 사람에 비해 RIF가 발생할 가능성이 낮다.1) When the miR-27a A> G genotype of the subject is the AG genotype, RIF is less likely to occur compared to the AA genotype (predominant genotype).

2) 피검자의 miR-449b A>G 유전자형이 AG 유전자형인 경우 AA 유전자형인 사람에 비해 3회 이상 또는 4회 이상의 RIF가 발생할 가능성이 높다.2) When the miR-449b A> G genotype of the test subject is the AG genotype, it is more likely that RIF occurs three or more times or more than four times compared to the AA genotype.

3) 피검자의 miR-604 A>G 유전자형이 GG 유전자형인 경우 AA 유전자형인 사람에 비해 RIF가 발생할 가능성이 낮다.3) When the miR-604 A> G genotype of the test subject is the GG genotype, the likelihood of RIF generation is lower than that of the AA genotype.

4) 피검자의 miR-27a A>G / miR-423 C>A 조합형이 AG/CC 조합형인 경우, AA/CC 조합형인 사람에 비해 RIF가 발생할 가능성이 낮다.4) When the subject's miR-27a A> G / miR-423 C> A combination type is the AG / CC combination type, it is less likely to develop RIF than the AA / CC combination type person.

5) 피검자의 miR-27a A>G / miR-604 A>G 조합형이 AA/GG 조합형, AG/AG 조합형 또는 AG/GG 조합형인 경우, AA/AA 조합형인 사람에 비해 RIF가 발생할 가능성이 낮다.5) When the miR-27a A> G / miR-604 A> G combination of subjects is AA / GG combination, AG / AG combination or AG / GG combination, RIF is less likely to occur compared to AA / AA combination. .

6) 피검자의 miR-423 C>A / miR-449b A>G 조합형이 CC/AG 조합형 또는 CA/GG 조합형인 경우, CC/AA 조합형인 사람에 비해 RIF가 발생할 가능성이 높다.6) When the subject's miR-423 C> A / miR-449b A> G combination type is CC / AG combination type or CA / GG combination type, RIF is more likely to occur than the CC / AA combination type.

7) 피검자의 miR-423 C>A / miR-604 A>G 조합형이 CC/AG 조합형인 경우, CC/AA 조합형인 사람에 비해 RIF가 발생할 가능성이 낮다.7) When the miR-423 C> A / miR-604 A> G combination type of the subject is the CC / AG combination type, it is less likely to develop RIF than the CC / AA combination type person.

8) 피검자의 miR-449b A>G / miR-604 A>G 조합형이 AG/AA 조합형인 경우, AA/AA 조합형인 사람에 비해 RIF가 발생할 가능성이 높다.8) When the subject's miR-449b A> G / miR-604 A> G combination type is the AG / AA combination type, RIF is more likely to occur than the AA / AA combination type.

9) 피검자의 miR-27a A>G / miR-423 C>A / miR-449b A>G / miR-604 A>G 일배체형이 A-C-G-A 일배체형, A-A-G-G 일배체형 또는 G-A-A-A 일배체형인 경우, A-C-A-A 일배체형인 사람에 비해 RIF가 발생할 가능성이 높다.9) If the subject's miR-27a A> G / miR-423 C> A / miR-449b A> G / miR-604 A> G haplotype is ACGA haplotype, AAGG haplotype or GAAA haplotype, ACAA fold RIFs are more likely to occur than people with body types.

10) 피검자의 miR-27a A>G / miR-423 C>A / miR-449b A>G / miR-604 A>G 일배체형이 G-C-A-G 일배체형인 경우, A-C-A-A 일배체형인 사람에 비해 RIF가 발생할 가능성이 낮다.10) If the subject's miR-27a A> G / miR-423 C> A / miR-449b A> G / miR-604 A> G haplotype is GCAG haplotype, RIF will occur compared to the ACAA haplotype. It is unlikely.

11) 피검자의 miR-27a A>G / miR-423 C>A / miR-449b A>G 일배체형이 G-C-A 일배체형인 경우, A-C-A 일배체형인 사람에 비해 RIF가 발생할 가능성이 낮다.11) When the subject's miR-27a A> G / miR-423 C> A / miR-449b A> G haplotype is G-C-A haplotype, it is less likely to develop RIF than the A-C-A haplotype.

12) 피검자의 miR-27a A>G / miR-423 C>A / miR-604 A>G 일배체형이 G-C-G 일배체형 또는 G-A-G 일배체형인 경우, A-C-A 일배체형인 사람에 비해 RIF가 발생할 가능성이 낮다.12) When the subject's miR-27a A> G / miR-423 C> A / miR-604 A> G haplotype is GCG haplotype or GAG haplotype, it is less likely to develop RIF than a person who is ACA haplotype. .

13) 피검자의 miR-27a A>G / miR-449b A>G / miR-604 A>G 일배체형이 A-G-A 일배체형인 경우, A-A-A 일배체형인 사람에 비해 RIF가 발생할 가능성이 높다.13) When the subject's miR-27a A> G / miR-449b A> G / miR-604 A> G haplotype is A-G-A haplotype, it is more likely to develop RIF than the A-A-A haplotype.

14) 피검자의 miR-27a A>G / miR-449b A>G / miR-604 A>G 일배체형이 G-A-G 일배체형인 경우, A-A-A 일배체형인 사람에 비해 RIF가 발생할 가능성이 낮다.14) When the subject's miR-27a A> G / miR-449b A> G / miR-604 A> G haplotype is G-A-G haplotype, it is less likely to develop RIF than the A-A-A haplotype.

15) 피검자의 miR-423 C>A / miR-449b A>G / miR-604 A>G 일배체형이 A-G-G 일배체형인 경우, C-A-A 일배체형인 사람에 비해 RIF가 발생할 가능성이 낮다.15) When the subject's miR-423 C> A / miR-449b A> G / miR-604 A> G haplotype is A-G-G haplotype, it is less likely to develop RIF than the C-A-A haplotype.

16) 피검자의 miR-27a A>G / miR-423 C>A 일배체형이 G-C 일배체형인 경우, A-C 일배체형인 사람에 비해 RIF가 발생할 가능성이 낮다.16) When the subject's miR-27a A> G / miR-423 C> A haplotype is G-C haplotype, it is less likely to develop RIF than the A-C haplotype.

17) 피검자의 miR-27a A>G / miR-604 A>G 일배체형이 G-G 일배체형인 경우, A-A 일배체형인 사람에 비해 RIF가 발생할 가능성이 낮다.17) If the subject's miR-27a A> G / miR-604 A> G haplotype is G-G haplotype, it is less likely to develop RIF than the A-A haplotype.

18) 피검자의 miR-423 C>A / miR-449b A>G 일배체형이 C-G 일배체형인 경우, C-A 일배체형인 사람에 비해 RIF가 발생할 가능성이 높다.18) When the subject's miR-423 C> A / miR-449b A> G haplotype is C-G haplotype, it is more likely to develop RIF than the C-A haplotype.

19) 피검자의 miR-449b A>G / miR-604 A>G 일배체형이 A-G 일배체형인 경우, A-A 일배체형인 사람에 비해 RIF가 발생할 가능성이 낮다.19) When the subject's miR-449b A> G / miR-604 A> G haplotype is A-G haplotype, it is less likely to develop RIF than the A-A haplotype.

20) 피검자의 miR-449b A>G 유전자형이 AG 또는 GG 유전자형이고 aPTT(activated partial thromboplastin time)가 29.4초 이하인 경우, miR-449b A>G 유전자형이 AA 유전자형이고 aPTT가 29.4초를 초과하는 사람에 비해 RIF가 발생할 가능성이 높다.20) If the subject's miR-449b A> G genotype is AG or GG genotype and the activated partial thromboplastin time (apPTT) is 29.4 seconds or less, miR-449b A> G genotype is AA genotype and aPTT exceeds 29.4 seconds. In comparison, RIF is more likely to occur.

본 발명의 한국 여성의 반복착상실패 발병 위험 예측용 키트는 상기와 같은 SNP 판별을 용이하게 수행할 수 있도록 하여 이를 바탕으로 한국 여성의 반복착상실패 발병 위험을 예측할 수 있도록 하는 것으로, SNP를 검출하는 수단으로는 SNP의 검출을 위해 통상적으로 사용되는 물질, 예를 들어 PCR용 프라이머, PCR-RFLP용 프라이머 및 제한효소, DNA-DNA hybridization용 프로브 등이 사용될 수 있다. 본 발명에서는 이중에서도 PCR-RFLP용 프라이머 및 제한효소를 검출 수단으로 하는 것이 바람직하며, 특히 위에서 언급한 SNP 판별용 프라이머 및 제한효소가 바람직하다.The kit for predicting the risk of repeated implantation failure of a Korean woman of the present invention is to enable easy identification of the SNP as described above, thereby predicting the risk of repeated implantation failure of a Korean woman. As a means, materials commonly used for the detection of SNPs, for example, primers for PCR, primers for PCR-RFLP and restriction enzymes, probes for DNA-DNA hybridization, and the like can be used. In the present invention, it is preferable to use PCR-RFLP primers and restriction enzymes as detection means, and particularly, the above-mentioned primers and restriction enzymes for SNP discrimination are preferred.

본 발명의 키트에는 상기와 같은 검출 수단 이외에도 SNP 검출에 사용되는 시약, 기구 등이 더 포함될 수 있다.In addition to the detection means as described above, the kit of the present invention may further include reagents, instruments, and the like used for SNP detection.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

[실시예][Example]

1. 방법1. Method

1-1. 연구 집단1-1. Study group

2010년 3월부터 2012년 12월 사이에 분당차병원(성남, 한국)의 불임치료센터 산부인과에서 RIF 환자를 모집하고, 120명의 RIF 환자와 219명의 대조군 참가자로부터 혈액샘플을 수집하였다. 모든 환자와 대조군 참가자는 한국인이다. 본 연구는 분당차병원의 심의위원회에 의해 승인되었고, 모든 환자로부터 사전동의를 받았다(reference no. PBC09-120).Between March 2010 and December 2012, RIF patients were recruited from the Obstetrics and Gynecology Center at the Bundang Cha Hospital (Seongnam, Korea), and blood samples were collected from 120 RIF patients and 219 control participants. All patients and control participants are Koreans. This study was approved by the deliberation committee of Bundang Cha Hospital and received prior consent from all patients (reference no. PBC09-120).

유산을 경험한 환자(임신 20주 미만)는 융모성 생식선 자극 호르몬(human chorionic gonadotropin)의 레벨 또는 초음파 및/또는 신체검사를 통해 확인하였다. 해부학적, 염색체적, 호르몬적, 감염적, 자가면역적 또는 혈전증적인 착상실패는 RIF를 진단할 때 일반적으로 적용되는 배제기준이므로 이를 진단받은 대상자는 연구 그룹에서 제외하였다. RIF 환자의 해부학적 이상은 초음파검사, 자궁난관조영술, 자궁경검사, 컴퓨터단층촬영 또는 자기공명영상법으로 확인하였다. 핵형분석은 표준 프로토콜을 사용하여 수행하였다. 고프로락틴혈증, 황체결핍, 갑상선질환을 포함한 호르몬적인 원인은 말초혈액에서 프로락틴, 갑상선자극호르몬, 유리 T4, 난포자극호르몬, 황체형성호르몬 및 프로게스테론의 레벨을 측정하여 평가하였다. 자가면역 질환 루푸스 및 항인지질증후군을 확인하기 위해 각각 루푸스 항응고인자 및 항카디오리핀 항체를 확인하였다. 혈전증적인 원인은 혈소판 감소증으로 규정하고, 단백질 C와 단백질 S의 결핍과 항-β2 당단백질 항체의 존재로 평가하였다. 본 연구에서 자궁기형이나 근본적인 의학적 질환으로 진단받은 피험자는 제외하였다. 또한, 배아학자가 IVF를 위한 배아의 질을 평가하였다. 대조그룹의 여성들은 분당차병원에서 모집되어 다음과 같은 등록기준을 충족하였다 : 정기적인 월경주기, 적어도 1번의 자연임신 경험, 유산의 경험 없음, 46, XX의 핵형.Patients who experienced abortion (less than 20 weeks of pregnancy) were identified by level or ultrasound and / or physical examination of chorionic gonadotropin. Anatomical, chromosomal, hormonal, infectious, autoimmune, or thrombotic implantation failures were excluded from the study group because they were the exclusion criteria generally applied when diagnosing RIF. Anatomical abnormalities of RIF patients were confirmed by ultrasonography, hysterosalpingography, hysteroscopy, computed tomography, or magnetic resonance imaging. Karyotyping was performed using standard protocols. Hormonal causes including hyperprolactinemia, luteal deficiency, and thyroid disease were evaluated by measuring the levels of prolactin, thyroid stimulating hormone, free T4, follicle stimulating hormone, luteinizing hormone and progesterone in peripheral blood. To confirm the autoimmune disease lupus and antiphospholipid syndrome, lupus anticoagulant and anticardiolipin antibodies were identified, respectively. The thrombotic cause was defined as thrombocytopenia, and was evaluated by the lack of protein C and protein S and the presence of anti-β2 glycoprotein antibody. In this study, subjects diagnosed with uterine malformations or underlying medical conditions were excluded. In addition, an embryonician assessed the quality of the embryo for IVF. Women in the control group were recruited from the Bundang Cha Hospital and met the following registration criteria: regular menstrual cycle, at least one natural pregnancy experience, no experience of miscarriage, karyotype 46, XX.

1-2. 호모시스테인, 엽산, 총콜레스테롤, 요산, 혈액요소질소, 크레아티닌 및 혈액응고상태의 평가1-2. Evaluation of homocysteine, folic acid, total cholesterol, uric acid, urea nitrogen, creatinine and blood coagulation status

12시간 금식 후 RIF 환자로부터 혈액 샘플을 수집하였다. 호모시스테인은 Abbott IMx 분석기(Abbott Laboratories, Abbott Park, IL, USA)를 사용한 형광편광 면역측정법을 통해 측정하였다. 엽산은 ACS 180 Plus 자동화 화학발광시스템(Bayer Diagnostics, Tarrytown, NY, USA)을 사용한 경쟁적 면역측정법을 통해 측정하였다. 총콜레스테롤, 요산, 혈액요소질소 및 크레아티닌은 상업적으로 이용가능한 효소비색테스트(Roche Diagnostics, GmbH, Mannheim, Germany)를 사용하여 측정하였다. 혈소판, 백혈구 및 헤모글로빈 수치는 Sysmex XE 2100 자동혈액분석시스템(Sysmex Corporation, Kobe, Japan)을 사용하여 얻었다. PT(prothrombin time) 및 aPTT(activated partial thromboplastin time)는 ACL TOP 자동 광-광학 응고계(LSI Medience, Tokyo, Japan)로 측정하였다.Blood samples were collected from RIF patients after 12 hours of fasting. Homocysteine was measured by fluorescence polarization immunoassay using an Abbott IMx analyzer (Abbott Laboratories, Abbott Park, IL, USA). Folic acid was measured by competitive immunoassay using an ACS 180 Plus automated chemiluminescence system (Bayer Diagnostics, Tarrytown, NY, USA). Total cholesterol, uric acid, blood urea nitrogen and creatinine were measured using commercially available enzyme colorimetric tests (Roche Diagnostics, GmbH, Mannheim, Germany). Platelet, leukocyte and hemoglobin levels were obtained using a Sysmex XE 2100 automated blood analysis system (Sysmex Corporation, Kobe, Japan). The prothrombin time (PT) and activated partial thromboplastin time (apTT) were measured with an ACL TOP automatic optical-optical coagulator (LSI Medience, Tokyo, Japan).

1-3. 호르몬 분석1-3. Hormone analysis

월경주기의 두 번째 또는 세 번째 날에 정맥천자로 여성의 혈액샘플을 채취하고, 측정 전에 혈액으로부터 혈청샘플을 수집하였다. 에스트라디올, 갑상선자극호르몬 및 프로락틴 레벨은 방사면역측정법(Beckman Coulter)으로 측정하였고, 난포자극호르몬 및 황체형성호르몬 레벨은 효소면역측정법(Siemens, Munich, Germany)을 사용하여 제조사의 지침에 따라 측정하였다.On the second or third day of the menstrual cycle, a female blood sample was collected by venipuncture, and a serum sample was collected from the blood before measurement. Estradiol, thyroid stimulating hormone and prolactin levels were measured by radioimmunoassay (Beckman Coulter), and follicle stimulating hormone and luteinizing hormone levels were measured according to manufacturer's instructions using enzyme immunoassay (Siemens, Munich, Germany). .

1-4. 유전형 분석1-4. Genotyping

G-DEX 혈액추출키트(인트론, 성남, 한국)를 사용하여 백혈구에서 게놈 DNA를 추출하였다. 모든 유전자 다형성은 분리된 게놈 DNA를 주형으로 사용한 PCR-RFLP 분석(95℃에서 30초간의 변성, 56~59℃에서 30초간의 어닐링 및 72℃에서 30초간의 연장을 35사이클)으로 결정하였다. miR-27a(rs895819) A>G에 대해서는 5'-GAA CTT AGC CAC TGT GAA CAC CAC TTG G-3'의 정방향 프라이머 및 5'-TTG CTT CCT GTC ACA AAT CAC ATT G-3'의 역방향 프라이머를 사용하고(어닐링 58℃), 182bp의 PCR 산물을 DraIII 제한효소로 처리하였다. miR-423(rs6505162) C>A에 대해서는 5'-GAA GCC CGA AGT TTG AGG GAG AAA C-3'의 정방향 프라이머 및 5'-CGG GGA GAA ACT CAA GCG CGC G-3'의 역방향 프라이머를 사용하고(어닐링 59℃), 144bp의 PCR 산물을 AciI 제한효소로 처리하였다. miR-449b(rs10061133) A>G에 대해서는 5'-GGT ATC CAG AGC ACT TCA TTG ACA-3'의 정??항 프라이머 및 5'-ACC TGA ATC AGG TAG GCA GTG TCT-3'의 역방향 프라이머를 사용하고(어닐링 56℃), 119bp의 PCR 산물을 BsmAI 제한효소로 처리하였다. miR-604(rs2368393) A>G에 대해서는 5'-CTT GGC TCA GTG GTC TGT TT-3'의 정방향 프라이머 및 5'-GTA CAG GGA CTG AAA GGT GAA G-3'의 역방향 프라이머를 사용하고(어닐링 59℃), 243bp의 PCR 산물을 BssSI 제한효소로 처리하였다. 각 제한효소(New England BioLabs, Beverly, MA, USA) 처리는 37℃에서 16시간 동안 수행하였다.Genomic DNA was extracted from white blood cells using a G-DEX blood extraction kit (Intron, Seongnam, Korea). All gene polymorphisms were determined by PCR-RFLP analysis using isolated genomic DNA as a template (denaturation at 95 ° C for 30 seconds, annealing at 56 to 59 ° C for 30 seconds, and extension at 30 ° C for 30 seconds for 35 cycles). For miR-27a (rs895819) A> G, 5'-GAA CTT AGC CAC TGT GAA CAC CAC TTG G-3 'forward primer and 5'-TTG CTT CCT GTC ACA AAT CAC ATT G-3' reverse primer Used (annealing 58 ° C.) and the 182 bp PCR product was treated with Dra III restriction enzyme. For miR-423 (rs6505162) C> A, a 5'-GAA GCC CGA AGT TTG AGG GAG AAA C-3 'forward primer and a 5'-CGG GGA GAA ACT CAA GCG CGC G-3' reverse primer were used. (Annealing 59 ° C.), a 144 bp PCR product was treated with Aci I restriction enzyme. For miR-449b (rs10061133) A> G, 5'-GGT ATC CAG AGC ACT TCA TTG ACA-3 'constant primer and 5'-ACC TGA ATC AGG TAG GCA GTG TCT-3' reverse primer Used (annealed 56 ° C), and the 119bp PCR product was treated with Bsm AI restriction enzyme. For miR-604 (rs2368393) A> G, 5'-CTT GGC TCA GTG GTC TGT TT-3 'forward primer and 5'-GTA CAG GGA CTG AAA GGT GAA G-3' reverse primer were used (annealing 59 ° C), 243 bp PCR product was treated with Bss SI restriction enzyme. Each restriction enzyme (New England BioLabs, Beverly, MA, USA) treatment was performed at 37 ° C. for 16 hours.

1-5. 통계 분석1-5. Statistical analysis

RIF 환자와 대조군의 유전자형 빈도는 Fisher exact test와 logistic regression을 사용하여 비교하였다. 모든 다형성은 Hardy-Weinberg 평형으로부터의 편차를 조사하기 위해 계산되었다. miRNA 다형성과 RIF 사이의 연관성 정도를 측정하기 위해 OR(odds ratio)와 95% CI(confidence interval)을 사용하였다. RIF 발병률과 연관된 다형성은 AOR(adjusted OR)과 나이를 보정한 logistic regression에서 95% CI를 사용하여 계산하였다. 각 특정 다형성과 대립유전자 조합 사이의 관계를 평가하기 위해서도 OR과 95% CI를 사용하였다. MDR(multifactor-dimensionality reduction) 방법과 MDR 소프트웨어 버전 2.0(www.epistasis.org)을 사용하여 4개의 miRNA 유전자좌 간의 유전자-유전자 상호작용을 분석하였다. 강한 시너지효과가 있는 조합을 결정하기 위해 다형성의 가능한 모든 조합을 MDR 분석을 사용하여 연구하였다. 통계적 분석은 GraphPad Prism 4.0(GraphPad Software Inc., San Diego, CA) 및 StatsDirect 소프트웨어 버전 2.4.4(StatsDirect Ltd., Altrincham, UK)를 사용하여 수행하였다. 유추된 유전자형과 RIF 사이의 유의적인 연관성은 사례 및 대조군 상태를 변경하고, 각 표본세트에 대해 1,000회 시험통계를 재계산하여 평가하였다.The genotyping frequency of RIF patients and controls was compared using Fisher exact test and logistic regression. All polymorphisms were calculated to investigate deviations from the Hardy-Weinberg equilibrium. OR (odds ratio) and 95% confidence interval (CI) were used to measure the degree of association between miRNA polymorphism and RIF. Polymorphism associated with RIF incidence was calculated using 95% CI in adjusted OR (AOR) and logistic regression corrected for age. OR and 95% CI were also used to evaluate the relationship between each specific polymorphism and allele combination. The gene-gene interaction between four miRNA loci was analyzed using the multifactor-dimensionality reduction (MDR) method and MDR software version 2.0 (www.epistasis.org). All possible combinations of polymorphism were studied using MDR analysis to determine combinations with strong synergies. Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA) and StatsDirect software version 2.4.4 (StatsDirect Ltd., Altrincham, UK). Significant association between the inferred genotype and RIF was evaluated by changing the case and control status, and recalculating 1,000 test statistics for each sample set.

2. 결과2. Results

RIF 환자군과 대조군의 임상적 특징은 표 1과 같다. RIF 환자는 aPTT가 현저히 낮고 에스트라디올과 황체형성호르몬이 높았다(모두 P<0.05). miRNA 유전자형(모두 Hardy-Weinberg 평형 성립)과 관련하여, miR-27aA>G 다형성(AOR, 0.480; 95% CI, 0.296-0.781; P=0.003 및 AOR, 0.499; 95% CI, 0.316-0.789; P=0.003)은 RIF 위험에 대한 보호효과를 보였다(표 2 참조). miR-604A>G(AOR, 0.415; 95% CI, 0.196-0.878; P=0.021 및 AOR, 0.596; 95% CI, 0.377-0.945; P=0.028) 다형성 또한 RIF 위험에 대한 보호효과를 보였으나, miR-449bA>G 다형성은 RIF 위험이 증가하는 것과 연관성이 있는 것으로 나타났다. 반복착상실패 횟수에 따라 RIF 환자를 계층화한 경우 3회 이상 실패한 환자에서 miR-27aA>G 다형성과 RIF 위험 사이에 연관성이 있었다.Table 1 shows the clinical characteristics of the RIF patient group and the control group. In RIF patients, aPTT was significantly lower and estradiol and luteinizing hormone were higher (all P <0.05). With respect to the miRNA genotype (all Hardy-Weinberg equilibrium established), miR-27aA> G polymorphism (AOR, 0.480; 95% CI, 0.296-0.781; P = 0.003 and AOR, 0.499; 95% CI, 0.316-0.789; P = 0.003) showed a protective effect against RIF risk (see Table 2). miR-604A> G (AOR, 0.415; 95% CI, 0.196-0.878; P = 0.021 and AOR, 0.596; 95% CI, 0.377-0.945; P = 0.028) polymorphism also showed protective effects against RIF risk, The miR-449bA> G polymorphism has been shown to be associated with increased RIF risk. When stratified RIF patients according to the number of repeated implantation failures, there was a correlation between miR-27aA> G polymorphism and RIF risk in patients who failed more than 3 times.

조합 분석 결과, miR-27aAG/miR-423CC 유전자형(AG/CC: AOR, 0.417; 95% CI, 0.223-0.780; P=0.006)을 갖는 환자와 miR-27aAG/miR-604AG 또는 GG 유전자형(AG/AG: AOR, 0.329; 95% CI, 0.153-0.706; P=0.004 및 AG/GG: AOR, 0.193; 95% CI, 0.054-0.693; P=0.012)을 갖는 환자에서 RIF 위험이 감소하는 것을 발견하였다(표 3 참조). 이러한 결과는 RIF 위험과 각 microRNA 유전자형 사이의 연관성과 일치한다.As a result of the combination analysis, patients with miR-27aAG / miR-423CC genotype (AG / CC: AOR, 0.417; 95% CI, 0.223-0.780; P = 0.006) and miR-27aAG / miR-604AG or GG genotype (AG / A decrease in RIF risk was found in patients with AG: AOR, 0.329; 95% CI, 0.153-0.706; P = 0.004 and AG / GG: AOR, 0.193; 95% CI, 0.054-0.693; P = 0.012). (See Table 3). These results are consistent with the association between RIF risk and each microRNA genotype.

네 가지 miRNA 다형성의 MDR 분석에 의한 대립유전자-대립유전자 조합의 결과는 표 4와 같으며, 여기에는 대립유전자 조합형과 RIF 유병률의 연관성에 대해서만 나타내었다. miR-27a/miR-423/miR-449b/miR-604에 대한 세 가지 일배체형(haplotype)이 RIF 위험 증가와 연관성이 있는 것으로 나타났다: A-C-G-A(OR, 2.352; 95% CI, 1.260-4.390; P=0.007), A-A-G-G(OR, 8.818; 95% CI, 1.004-77.460; P=0.030), 및 G-A-A-A(OR, 5.291; 95% CI, 1.627-17.210; P=0.006); G-C-A-G(OR, 0.248; 95% CI, 0.115-0.537; P=0.0001) 일배체형은 위험 감소와 연관이 있는 것으로 나타났다. RIF의 높은 위험도는 또한 miR-27a/miR-449b/miR-604의 A-G-A 일배체형(OR, 1.933; 95% CI, 1.146-3.261; P=0.016)과 연관성이 있는 것으로 나타났다. 이와 반대로, miR-27a/miR-449b/miR-604의 G-A-G 및 miR-423/miR-449b/miR-604의 A-G-G는 RIF 위험 감소와 연관성이 있는 것으로 나타났다.The results of the allele-allele combination by MDR analysis of the four miRNA polymorphisms are shown in Table 4. Here, only the association between the allele combination and RIF prevalence was shown. Three haplotypes for miR-27a / miR-423 / miR-449b / miR-604 have been shown to be associated with increased RIF risk: ACGA (OR, 2.352; 95% CI, 1.260-4.390; P = 0.007), AAGG (OR, 8.818; 95% CI, 1.004-77.460; P = 0.030), and GAAA (OR, 5.291; 95% CI, 1.627-17.210; P = 0.006); GCAG (OR, 0.248; 95% CI, 0.115-0.537; P = 0.0001) haplotype was shown to be associated with reduced risk. The high risk of RIF has also been shown to be correlated with the AGA haplotype of miR-27a / miR-449b / miR-604 (OR, 1.933; 95% CI, 1.146-3.261; P = 0.016). In contrast, the GAG of miR-27a / miR-449b / miR-604 and the AGG of miR-423 / miR-449b / miR-604 have been shown to be associated with reduced RIF risk.

네 가지 miRNA 다형성과 환경적 요인 사이의 상호작용 연관성에서 RIF의 유병률을 분석하였을 때, miR-27aAG+GG 유전자형은 PT가 10.45초 이하인 환자에서 RIF와 연관성이 있는 것으로 나타났다(AOR, 0.058; 95% CI, 0.015-0.251; 표 5 참조). 또한 miR-423CA+AA 유전자형은 PT가 10.45초 이하(AOR, 0.003; 95% CI, 0.000-0.047), aPTT가 29.4초 이하(AOR, 0.188; 95% CI, 0.039-0.904), 혈소판수가 276x103/㎕ 이상(AOR, 0.041; 95% CI, 0.012-0.0140)인 환자에서 RIF와 연관성이 있는 것으로 나타났다. miR-449bAG 또는 GG 유전자형은 PT가 10.45초 이하(AOR, 0.166; 95% CI, 0.042-0.653), aPTT가 29.4초 이하(AOR, 7.980; 95% CI, 1.998-31.870), 및 혈소판수가 276x103/㎕ 이상(AOR, 2.831; 95% CI, 1.123-7.138)인 환자에서 RIF와 연관성이 있는 것으로 나타났다. miR-604AG 또는 GG 유전자형은 PT가 10.45초 이하(AOR, 0.080; 95% CI, 0.018-0.351)인 환자에서 RIF와 연관성이 있는 것으로 나타났다. 추가적으로, 의미있는 각 유전자형 및 일배체형 조합 그룹의 통계적인 타당성을 확인하고자 통계력 분석을 수행하였다(표 6 참조).When analyzing the prevalence of RIF in the interaction relationship between four miRNA polymorphisms and environmental factors, the miR-27aAG + GG genotype was found to be associated with RIF in patients with PTs less than 10.45 seconds (AOR, 0.058; 95%) CI, 0.015-0.251; see Table 5). In addition, miR-423CA + AA genotype has PT of 10.45 seconds or less (AOR, 0.003; 95% CI, 0.000-0.047), aPTT of 29.4 seconds or less (AOR, 0.188; 95% CI, 0.039-0.904), and platelet count is 276x10 3 It was found to be related to RIF in patients with / µl or more (AOR, 0.041; 95% CI, 0.012-0.0140). For miR-449bAG or GG genotype, PT is 10.45 seconds or less (AOR, 0.166; 95% CI, 0.042-0.653), aPTT is 29.4 seconds or less (AOR, 7.980; 95% CI, 1.998-31.870), and platelet count is 276x10 3 It was found to be related to RIF in patients with / µl or higher (AOR, 2.831; 95% CI, 1.123-7.138). The miR-604AG or GG genotype has been shown to be associated with RIF in patients with PTs less than 10.45 seconds (AOR, 0.080; 95% CI, 0.018-0.351). Additionally, statistical analysis was performed to confirm the statistical validity of each meaningful genotype and haplotype combination group (see Table 6).

<표 설명><Table description>

표 1 : SD, 표준편차; BMI = body mass index; PT, prothrombin; aPTT, Activated Partial Thromboplastin Time; Hcy, Homocysteine; Hgb, hemoglobin; PLT, platelet count; BUN, blood urea nitrogen; TSH, thyroid-stimulating hormone; FSH, follicle stimulating hormone; LH, luteinizing hormone; NA = not applicable; RIF = recurrent implantation failure.Table 1: SD, standard deviation; BMI = body mass index; PT, prothrombin; aPTT, Activated Partial Thromboplastin Time; Hcy, Homocysteine; Hgb, hemoglobin; PLT, platelet count; BUN, blood urea nitrogen; TSH, thyroid-stimulating hormone; FSH, follicle stimulating hormone; LH, luteinizing hormone; NA = not applicable; RIF = recurrent implantation failure.

표 2 : AOR은 참가자의 연령으로 조정됨; RIF = recurrent implantation failure; AOR = adjusted odds ratio; CI = confidence interval.Table 2: AOR adjusted to age of participants; RIF = recurrent implantation failure; AOR = adjusted odds ratio; CI = confidence interval.

표 3 : RIF = recurrent implantation failure; AOR = adjusted odds ratio; CI = confidence interval.Table 3: RIF = recurrent implantation failure; AOR = adjusted odds ratio; CI = confidence interval.

표 4 : RIF = recurrent implantation failure; 각 대립유전자 조합의 OR과 95% CI는 Fisher's exact test를 사용하여 모든 다른 대립유전자의 빈도를 참고하여 계산됨; Fisher's exact test에 따른 P값; OR = odds ratio; CI = confidence interval.Table 4: RIF = recurrent implantation failure; OR and 95% CI of each allele combination was calculated using Fisher's exact test to reference the frequencies of all other alleles; P value according to Fisher's exact test; OR = odds ratio; CI = confidence interval.

표 5 : RIF = recurrent implantation failure; *PT 10.45초, aPTT 29.4초는 RIF 환자 및 대조군에서 하위 25% cut off 레벨; †Platelet 276 103/㎕는 RIF 환자 및 대조군에서 상위 25% cut off 레벨.Table 5: RIF = recurrent implantation failure; * PT 10.45 sec, aPTT 29.4 sec in the lower 25% cut off level in RIF patients and controls; † Platelet 276 103 / μl is the top 25% cut off level in RIF patients and controls.

표 6 : AOR = adjusted odds ratio; CI = confidence interval.Table 6: AOR = adjusted odds ratio; CI = confidence interval.

Figure pat00001
Figure pat00001

Figure pat00002
Figure pat00002

Figure pat00003
Figure pat00003

Figure pat00004
Figure pat00004

Figure pat00005
Figure pat00005

Figure pat00006
Figure pat00006

<110> CHA University Industry-Academic Cooperation Foundation <120> Association of miR-27a A>G, miR-423 C>A, miR-449b A>G and miR-604 A>G single nucleotide polymorphism with the risk of recurrent implantation failure in a Korean women <130> PA-D18256 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 321 <212> DNA <213> Homo sapiens <400> 1 gccggctggg gttcctgggg atgggatttg cttcctgtca caaatcacat tgccagggat 60 ttccaaccga ccctgagctc tgccaccgag gatgctgccc ggggacgggg tggcagagag 120 gccccgaagc ctgtgcctgg cctgaggagc agggcttagc tgcttgtgag cagggtccac 180 rccaagtcgt gttcacagtg gctaagttcc gccccccagg ccctcacctc ctctggcctt 240 gccgcctgtc ccctgctgcc gcctgtctgc ctgccatcct gctgcctggc ctccctgggc 300 tctgcctccc gtgcctactg a 321 <210> 2 <211> 321 <212> DNA <213> Homo sapiens <400> 2 ccgtacattt tcccggatgg aagcccgaag tttgagggag aaacttgtga ggaaataaag 60 gaagttaggc tgaggggcag agagcgagac ttttctattt tccaaaagct cggtctgagg 120 cccctcagtc ttgcttccta mcccgcgctt gagtttctcc ccgcttggat gctctcaggg 180 gcagtgtgaa gagagacagt tcctggcttc cttagagggc cttgttctcc ttggcagaag 240 ctcaagtcct gattcccgcc cttccttctc gttttaggaa tattgttctg agaagccaca 300 gaaaaagggt aggcatatag g 321 <210> 3 <211> 321 <212> DNA <213> Homo sapiens <400> 3 cacatattca accagctaac aatacactgc cagctcatca cacacaggta tccagagcac 60 ttcattgaca agaagaattt atccagaagc aagtggcagg gtagttgtgg ctgctgactt 120 gacccaagca gccagctaac ratacactgc ctacctgatt caggtcactg ccgattctcg 180 tggcccaggc agtcactgag gctaaatttg taacatgaaa catagtggca gaactctaat 240 cttaatagtc acattaaaga aaacttatac acaaaatgta ttacttcaag actataagaa 300 ataaaatcaa tatttgcttg a 321 <210> 4 <211> 321 <212> DNA <213> Homo sapiens <400> 4 aggtgtttgg gggtacaggg actgaaaggt gaagccaatt ccattccagt aatagaataa 60 gctgtgagga ggacccagat gtctgttctt tagaatggcc cccaagatgt atgtttctcc 120 atgatgcact gtcctgaatt ccgcagcctg tgtcagaaaa cctgcctgct agtggacacg 180 rgagcgtgga aggtcaagca cgatgctctc ctgcagacac ctctctcagt gcatggaaac 240 agaccactga gccaagagag cttgaggctc tggagacggt ttttagacta aaatctctca 300 tcaggagatg agcaaatgca a 321 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-27a A>G <400> 5 gaacttagcc actgtgaaca ccacttgg 28 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-27a A>G <400> 6 ttgcttcctg tcacaaatca cattg 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-423 C>A <400> 7 gaagcccgaa gtttgaggga gaaac 25 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-423 C>A <400> 8 cggggagaaa ctcaagcgcg cg 22 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-449b A>G <400> 9 ggtatccaga gcacttcatt gaca 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-449b A>G <400> 10 acctgaatca ggtaggcagt gtct 24 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-604 A>G <400> 11 cttggctcag tggtctgttt 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-604 A>G <400> 12 gtacagggac tgaaaggtga ag 22 <110> CHA University Industry-Academic Cooperation Foundation <120> Association of miR-27a A> G, miR-423 C> A, miR-449b A> G and miR-604          A> G single nucleotide polymorphism with the risk of recurrent          implantation failure in a Korean women <130> PA-D18256 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 321 <212> DNA <213> Homo sapiens <400> 1 gccggctggg gttcctgggg atgggatttg cttcctgtca caaatcacat tgccagggat 60 ttccaaccga ccctgagctc tgccaccgag gatgctgccc ggggacgggg tggcagagag 120 gccccgaagc ctgtgcctgg cctgaggagc agggcttagc tgcttgtgag cagggtccac 180 rccaagtcgt gttcacagtg gctaagttcc gccccccagg ccctcacctc ctctggcctt 240 gccgcctgtc ccctgctgcc gcctgtctgc ctgccatcct gctgcctggc ctccctgggc 300 tctgcctccc gtgcctactg a 321 <210> 2 <211> 321 <212> DNA <213> Homo sapiens <400> 2 ccgtacattt tcccggatgg aagcccgaag tttgagggag aaacttgtga ggaaataaag 60 gaagttaggc tgaggggcag agagcgagac ttttctattt tccaaaagct cggtctgagg 120 cccctcagtc ttgcttccta mcccgcgctt gagtttctcc ccgcttggat gctctcaggg 180 gcagtgtgaa gagagacagt tcctggcttc cttagagggc cttgttctcc ttggcagaag 240 ctcaagtcct gattcccgcc cttccttctc gttttaggaa tattgttctg agaagccaca 300 gaaaaagggt aggcatatag g 321 <210> 3 <211> 321 <212> DNA <213> Homo sapiens <400> 3 cacatattca accagctaac aatacactgc cagctcatca cacacaggta tccagagcac 60 ttcattgaca agaagaattt atccagaagc aagtggcagg gtagttgtgg ctgctgactt 120 gacccaagca gccagctaac ratacactgc ctacctgatt caggtcactg ccgattctcg 180 tggcccaggc agtcactgag gctaaatttg taacatgaaa catagtggca gaactctaat 240 cttaatagtc acattaaaga aaacttatac acaaaatgta ttacttcaag actataagaa 300 ataaaatcaa tatttgcttg a 321 <210> 4 <211> 321 <212> DNA <213> Homo sapiens <400> 4 aggtgtttgg gggtacaggg actgaaaggt gaagccaatt ccattccagt aatagaataa 60 gctgtgagga ggacccagat gtctgttctt tagaatggcc cccaagatgt atgtttctcc 120 atgatgcact gtcctgaatt ccgcagcctg tgtcagaaaa cctgcctgct agtggacacg 180 rgagcgtgga aggtcaagca cgatgctctc ctgcagacac ctctctcagt gcatggaaac 240 agaccactga gccaagagag cttgaggctc tggagacggt ttttagacta aaatctctca 300 tcaggagatg agcaaatgca a 321 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-27a A> G <400> 5 gaacttagcc actgtgaaca ccacttgg 28 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-27a A> G <400> 6 ttgcttcctg tcacaaatca cattg 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-423 C> A <400> 7 gaagcccgaa gtttgaggga gaaac 25 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-423 C> A <400> 8 cggggagaaa ctcaagcgcg cg 22 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-449b A> G <400> 9 ggtatccaga gcacttcatt gaca 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-449b A> G <400> 10 acctgaatca ggtaggcagt gtct 24 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-604 A> G <400> 11 cttggctcag tggtctgttt 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-604 A> G <400> 12 gtacagggac tgaaaggtga ag 22

Claims (6)

한국 여성의 반복착상실패 발병 위험 예측에 필요한 정보를 제공하기 위하여 피검자의 DNA 시료로부터 miR-27a A>G(rs895819), miR-449b A>G(rs10061133) 및 miR-604 A>G(rs2368393) 중에서 선택된 단일염기다형성을 판별하는 방법.MiR-27a A> G (rs895819), miR-449b A> G (rs10061133) and miR-604 A> G (rs2368393) from DNA samples of subjects to provide the information necessary to predict the risk of repeated implantation failure in Korean women A method of determining a single base polymorphism selected from among. 제 1항에 있어서,
miR-423 C>A(rs6505162) 단일염기다형성을 더 판별하는 것을 특징으로 하는 방법.
According to claim 1,
miR-423 C> A (rs6505162) A method characterized by further discriminating monobasic polymorphism.
miR-27a A>G(rs895819), miR-449b A>G(rs10061133) 및 miR-604 A>G(rs2368393) 중에서 선택된 단일염기다형성을 검출하는 수단을 포함하는 한국 여성의 반복착상실패 발병 위험 예측용 키트.Predicting the risk of repeated implantation failure in Korean women, including means for detecting single-base polymorphism selected from miR-27a A> G (rs895819), miR-449b A> G (rs10061133) and miR-604 A> G (rs2368393) Dragon kit. 제 3항에 있어서,
miR-423 C>A(rs6505162) 단일염기다형성을 검출하는 수단을 더 포함하는 것을 특징으로 하는 키트.
According to claim 3,
kit further comprising means for detecting miR-423 C> A (rs6505162) monobasic polymorphism.
제 3항에 있어서,
상기 miR-27a A>G 단일염기다형성을 검출하는 수단은 서열번호 5의 염기서열로 표시되는 올리고뉴클레오티드 프라이머, 서열번호 6의 염기서열로 표시되는 올리고뉴클레오티드 프라이머 및 DraIII 제한효소를 포함하여 이루어지는 프라이머-제한효소 세트이고,
상기 miR-449b A>G 단일염기다형성을 검출하는 수단은 서열번호 9의 염기서열로 표시되는 올리고뉴클레오티드 프라이머, 서열번호 10의 염기서열로 표시되는 올리고뉴클레오티드 프라이머 및 BsmAI 제한효소를 포함하여 이루어지는 프라이머-제한효소 세트이고,
상기 miR-604 A>G 단일염기다형성을 검출하는 수단은 서열번호 11의 염기서열로 표시되는 올리고뉴클레오티드 프라이머, 서열번호 12의 염기서열로 표시되는 올리고뉴클레오티드 프라이머 및 BssSI 제한효소를 포함하여 이루어지는 프라이머-제한효소 세트인 것을 특징으로 하는 키트.
According to claim 3,
The means for detecting the miR-27a A> G single base polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 5, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 6, and a primer comprising a Dra III restriction enzyme -Restriction enzyme set,
The means for detecting the miR-449b A> G single base polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 9, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 10, and a primer comprising Bsm AI restriction enzyme. -Restriction enzyme set,
The means for detecting the miR-604 A> G single base polymorphism is a oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 11, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 12, and a primer comprising Bss SI restriction enzyme -A kit characterized by being a restriction enzyme set.
제 4항에 있어서,
상기 miR-423 C>A 단일염기다형성을 검출하는 수단은 서열번호 7의 염기서열로 표시되는 올리고뉴클레오티드 프라이머, 서열번호 8의 염기서열로 표시되는 올리고뉴클레오티드 프라이머 및 AciI 제한효소를 포함하여 이루어지는 프라이머-제한효소 세트인 것을 특징으로 하는 키트.
The method of claim 4,
The means for detecting the miR-423 C> A single base polymorphism is a oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 7, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 8, and a primer comprising an Aci I restriction enzyme -A kit characterized by being a restriction enzyme set.
KR1020180143428A 2018-11-20 2018-11-20 Association of miR―27a A>G, miR―423 C>A, miR―449b A>G and miR―604 A>G single nucleotide polymorphism with the risk of recurrent implantation failure in a Korean women KR102143770B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020180143428A KR102143770B1 (en) 2018-11-20 2018-11-20 Association of miR―27a A>G, miR―423 C>A, miR―449b A>G and miR―604 A>G single nucleotide polymorphism with the risk of recurrent implantation failure in a Korean women

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020180143428A KR102143770B1 (en) 2018-11-20 2018-11-20 Association of miR―27a A>G, miR―423 C>A, miR―449b A>G and miR―604 A>G single nucleotide polymorphism with the risk of recurrent implantation failure in a Korean women

Publications (2)

Publication Number Publication Date
KR20200058832A true KR20200058832A (en) 2020-05-28
KR102143770B1 KR102143770B1 (en) 2020-08-12

Family

ID=70920032

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020180143428A KR102143770B1 (en) 2018-11-20 2018-11-20 Association of miR―27a A>G, miR―423 C>A, miR―449b A>G and miR―604 A>G single nucleotide polymorphism with the risk of recurrent implantation failure in a Korean women

Country Status (1)

Country Link
KR (1) KR102143770B1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20100070776A (en) * 2008-12-18 2010-06-28 차의과학대학교 산학협력단 Biomarkers for the diagnosis of recurrent pregnancy loss
US20110229894A1 (en) * 2008-11-30 2011-09-22 Rosetta Genomics Ltd. Methods for detecting an increased susceptibility to cancer
KR101169653B1 (en) * 2011-12-09 2012-08-03 의료법인 성광의료재단 Method of providing information for diagnosis of idiopathic recurrent spontaneous abortion using microrna polymorphism and diagnostic composition
US20180105878A1 (en) * 2016-09-14 2018-04-19 National Sun Yat-Sen University Biomarker of detecting a biological sample, probe, kit and method of non-invasively and qualitatively determining severity of endometriosis

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110229894A1 (en) * 2008-11-30 2011-09-22 Rosetta Genomics Ltd. Methods for detecting an increased susceptibility to cancer
KR20100070776A (en) * 2008-12-18 2010-06-28 차의과학대학교 산학협력단 Biomarkers for the diagnosis of recurrent pregnancy loss
KR101169653B1 (en) * 2011-12-09 2012-08-03 의료법인 성광의료재단 Method of providing information for diagnosis of idiopathic recurrent spontaneous abortion using microrna polymorphism and diagnostic composition
US20180105878A1 (en) * 2016-09-14 2018-04-19 National Sun Yat-Sen University Biomarker of detecting a biological sample, probe, kit and method of non-invasively and qualitatively determining severity of endometriosis

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
European Journal of Obstetrics & Gynecology and Reproductive Biology (2016) 202:14-19 *
PLoS ONE (2017.05.10.) 12(5):e0177160* *
Reprod Biomed Online. 2014 Jan;28(1):14-38.
SAKONG 등, "Association Study between miRNA (miR-27a, miR-423, miR-449b and miR-604) Polymorphisms and Recurrent Implantation Failure in Korean Women", 제71회 한국생물과학협회 정기학술대회, 포스터 발표 G553 (2016.08.10.-11* *
장효근, Association of the miR-604A>G, miR-608C>G, miR-631I/D, miR-938C>T, and miR-1302-3C>T polymorphisms with risk of idiopathic recurrent pregnancyloss in Korean women, 석사학위논문, 차의과학대학교 (2015.02.)* *

Also Published As

Publication number Publication date
KR102143770B1 (en) 2020-08-12

Similar Documents

Publication Publication Date Title
US20200208225A1 (en) Systems for detecting dna orginating from different individuals
EP2419526B1 (en) Methods for selecting oocytes and competent embryos with high potential for pregnancy outcome
US10947593B2 (en) Biomarkers for premature birth
AU731856B2 (en) Methods of screening for ulcerative colitis by detecting an interleukin-1 receptor antagonist polymorphism
Lim et al. Cell-free fetal DNA and cell-free total DNA levels in spontaneous abortion with fetal chromosomal aneuploidy
JP2003516161A (en) Methods for nucleic acid detection
US9090938B2 (en) Methods for selecting competent oocytes and competent embryos with high potential for pregnancy outcome
WO2009143576A1 (en) Polymorphisms associated with pregnancy complications
KR101256206B1 (en) An analysis method for determining the fetal gender and apparatus therefor
KR102143770B1 (en) Association of miR―27a A&gt;G, miR―423 C&gt;A, miR―449b A&gt;G and miR―604 A&gt;G single nucleotide polymorphism with the risk of recurrent implantation failure in a Korean women
US20200071762A1 (en) Method for predicting the risk of deep vein thrombosis and pulmonary embolism associated with hormonal preparations and hormone levels
CN111944893B (en) MiRNA molecular marker related to prenatal noninvasive diagnosis of cleft lip and palate and application thereof
KR102113061B1 (en) Association of miR-605 A&gt;G, miR-608 G&gt;C, miR-631 I&gt;D, miR-938 C&gt;T and miR-1302-3 C&gt;T polymorphism with the risk of recurrent implantation failure in a Korean women
JP4889258B2 (en) Method for determining resistance to the onset of bovine leukemia
WO2020119626A1 (en) Method for non-invasive prenatal testing of fetus for genetic disease
JP2010187556A (en) Pregnancy-diagnosing method, pregnancy-diagnosing kit, polynucleotide, polypeptide, and antibody
KR20150032062A (en) Fetal Epigenetic Markers and Use thereof
RU2719411C1 (en) Method for determining predisposition to reproductive disorders in females in conditions of excessive phenol contamination
US6268145B1 (en) Screening test for the lethal genetic trait of recurrent spontaneous pregnancy loss
EP3333267A1 (en) Method and kit for predicting the risk of deep vein thrombosis and pulmonary embolism
KR101879498B1 (en) Compositions for diagnosis of hypertensive disorders of pregnancy comprising biomarkers
KR101546366B1 (en) Composition for detecing fetal epigenetic markers and detecting method thereof
Brunet et al. The Progress of Next Generation Sequencing in Preimplantation Genetic Testing
JP2011239750A (en) Method for examining zdbf2 methylation failure as index of imprint failure
CN111979309A (en) Combined set for detecting trisomy syndrome

Legal Events

Date Code Title Description
E701 Decision to grant or registration of patent right
GRNT Written decision to grant