KR101695347B1 - Composition for predicting a risk of preterm delivery using CpG methylation status of gene and uses thereof - Google Patents

Abstract

The present invention relates to a composition for diagnosing the risk of premature birth by detecting the level of methylation on CpG regions of genes in a specimen of a pregnant patient. The present invention further relates to a diagnostic kit and a diagnostic method for diagnosing the risk of premature birth. To this end, the composition includes at least one preparation selected from the group consisting of microRNA-886 (mir-886) and INS-IGF2 readthrough for measuring the level of methylation on the CpG regions of genes of the patient.

Description

[0001] The present invention relates to a composition for predicting preterm birth using a CpG methylation change in a gene and a method for predicting preterm delivery using CpG methylation,

The present invention relates to a composition, a diagnostic kit and a diagnostic method for diagnosing the risk of premature birth by detecting the methylation level of a CpG region of a gene from a maternal sample.

Preterm birth usually means giving birth before 37 weeks after 20 weeks. The risk of death is high enough to account for about 60% of all infant deaths, and neonatal intensive care is needed for neonatal developmental disorders, respiratory complications, postnatal growth retardation, There is a high morbidity rate for long-term or short-term diseases. In addition, about 75% of preterm birth occurs with premature labor, amniocentesis, and associated cervical incompetence and chorioamnionitis. However, the mechanism associated with preterm delivery is still unclear. Common risk factors for natural labor include genital tract infections, multiple pregnancies, a few thirds of bleeding, and previous preterm delivery history.

The minimum gestational age is 27 weeks and the birth weight is 0.9 kg. The morbidity and mortality of the newborn are known to be influenced primarily by gestational age, or maturity, rather than birth weight. Thus, delaying the delivery of newborns by appropriate treatment to raise maturity when early signs of premature birth come, is a crucial issue for the health and quality of life and costs of mothers and newborns.

Until now, for the treatment of premature labor, maternal use of ant contractions, antibiotics, steroids and progesterone have been used to suppress labor and delay delivery. However, in cases of preterm labor and premature rupture of membranes, the treatment effect of antibiotics on intrauterine infection and inflammation is very limited and it is known that preterm delivery can not be prevented. In addition, women with cervical incompetence have been given cervical cerclage as a treatment and prophylaxis because of the great risk of infection and miscarriage, but this can not be treated as a fundamental treatment, It is known.

Therefore, it is necessary to select maternal risk pregnancies by anticipating the risk of premature birth, to prevent premature birth through appropriate management of them, or to develop an effective treatment method. In addition, the earlier the preterm delivery period, the more likely it is to leave a sequel to the neonate and the degree of sequelae is so severe that it is expected that the incidence of preterm infants and children with disabilities can be significantly reduced if the preterm delivery can be predicted.

A common approach to predicting preterm birth was to identify female populations that should be given special attention in terms of obstetric, demographic and various syndromes (Main et al., Am. J. Obste. Gynecol. , 151: 892-898, 1985), this approach has the problem that the method is not sensitive or specific. To overcome these problems, many studies have been conducted to find biochemical markers for predicting sudden preterm delivery and amniotic membrane wasting. Plasma estradiol-17 beta, progesterone, C Substances such as C-reactive protein have been nominated, but this has also been shown to be less accurate. Therefore, it is necessary to develop a marker that is easier to detect and has high sensitivity and specificity.

Korean Patent Laid-Open Publication No. 2002-0039458 (May 27, 2002)

Main et al., Am. J. Obste. Gynecol., 151: 892-898 (1985)

Accordingly, the present invention aims to contribute to the prevention of premature birth by identifying predictive markers of preterm delivery indicative of prematurity risk in the mother.

For this purpose, the object of the present invention is to provide a method for measuring the methylation level of the CpG region of one or more genes selected from the group consisting of mir-886 (microRNA-886) and INS-IGF2 (INS-IGF2 readthrough) And a method for diagnosing maternal risk of premature birth.

As another example, another object of the present invention is to provide a kit for the diagnosis of maternal risk of premature birth comprising the composition.

As another example, another object of the present invention is to provide a method of detecting methylation level of a CpG region of at least one gene selected from the group consisting of mir-886 and INS-IGF2 from a maternal sample; And comparing the methylation level to the methylation level of the CpG region of the gene of the normal delivery mother.

As another example, another object of the present invention is to provide a method of detecting methylation level of a CpG region of at least one gene selected from the group consisting of mir-886 and INS-IGF2 from a maternal sample; And comparing said methylation level with a methylation level of a CpG region of a gene of normal delivery mother.

The present invention uses the degree of methylation of the CpG region of the above genes as a biomarker based on the results of selecting genes showing differences in the level of CpG methylation in the blood between the premature delivery mother and the normal delivery mother To provide techniques for predicting preterm birth risk

In one embodiment of the present invention, blood was collected from pregnant women who were admitted to the premature labor during pregnancy, and the fetuses were identified to distinguish between the premature births of less than 37 weeks and the normal births after 37 weeks. . Genome-Wide DNA Methylation Patterns were also analyzed using Illumina HumanMethylation 450 BeadChip (Illumina) to compare the methylation level of genes. The genes with statistically significant differences in DNA methylation levels were selected in the premature maternal group. Furthermore, we identified specific CpG sites that affected gene expression and confirmed that the risk of preterm birth could be predicted by measuring the degree of methylation occurring at specific CpG sites of the gene.

Thus, hypermethylation or hypomethylation of DNA methylation at the CpG site of the mir-886 and / or INS-IGF2 gene can be used as a biomarker to diagnose premature birth or predict preterm delivery risk .

More specifically, the CpG region of mir-886 is specifically hypermethylated in preterm premature rats and the CpG region of the INS-IGF2 gene is specifically hypomethylated in prematurity-risk mothers. May be used as a single biomarker for predicting premature rupture risk, or two or more of them may be used as a complex biomarker.

Accordingly, in one aspect, the invention provides a diagnostic composition for the diagnosis of premature rupture, comprising a composition for measuring the methylation level of the CpG region of at least one gene selected from the group consisting of mir-886 and INS-IGF2, Kit.

The term "diagnosis" or "prediction" in the present invention means that the mother is likely to undergo preterm birth in less than 37 weeks of gestation, is relatively more likely to have preterm birth in less than 37 weeks, And the like. The present invention may be used to delay or prevent premature delivery of maternal mothers who are at high risk of premature birth for less than 37 weeks through special and appropriate care. In addition, the invention can be used clinically to make treatment decisions by early diagnosis of preterm birth of less than 37 weeks and by selecting the most appropriate treatment regimen.

The term "diagnostic markers, diagnostic markers or diagnostic markers" or "markers" in the present invention refers to preterm birth mothers (for example, And maternal births of more than a week), which means bioorganic molecules that show significant differences in maternal samples predicted by preterm delivery compared to normal maternal maternal samples. For purposes of the present invention, it may refer to genes that specifically show DNA methylation changes in maternal samples predicted by premature birth.

 In the present invention, the term "methylation" refers to the attachment of a methyl group to a base constituting DNA. Preferably, the methylation in the present invention means whether methylation occurs in the cytosine of a specific CpG site of a specific gene. When methylation occurs, the binding of transcription factors is hindered and the expression of a specific gene is inhibited. On the other hand, when unmethylated or hypomethylated, the expression of a specific gene is increased.

Genomic DNA of mammalian cells contains 5th methyl-cytosine (5-methylcytosine, 5-mC) base with a methyl group attached to the fifth carbon of the cytosine ring in addition to A, C, G and T do. Methylation of 5-methylcytosine occurs only in C of a CG dinucleotide (5'-mCG-3 ') called CpG, and methylation of CpG inhibits expression of repeated sequences of alu or transposon and genome. Also, since 5-mC of the CpG is naturally deaminated and is likely to become a thymine (T), CpG is a site where most epigenetic changes frequently occur in mammalian cells.

The term "measurement of methylation level" in the present invention means the measurement of the methylation level of the CpG region of the mir-886 and / or INS-IGF2 gene by methylation-specific PCR, for example methylation- chain reaction, MSP), real-time methylation-specific polymerase chain reaction (PCR), PCR using methylated DNA-specific binding protein, or quantitative PCR. Or by automated base analysis such as pyrosequencing and bisulfite sequencing, but are not limited thereto.

Preferably, the CpG region of the gene refers to a CpG region present on the DNA of the gene. The DNA of the gene includes a promoter region, an open reading frame (ORF), and a terminator (ORF), all of which are necessary to express the gene and include a series of constituent units operably linked to each other. Region. Thus, the CpG site of the mir-886 and / or INS-IGF2 gene may be present in the promoter region, protein coding region (ORF) or terminator region of the gene of interest. A preferred example may be a CpG site present in the promoter region of the gene.

Preferably, measuring the methylation level of the CpG region of mir-886 in the present invention may mean measuring the methylation level of the cytosine of the CpG region present in the 135416005 to 135416405 base of chromosome 5. In the present invention, nucleotides 135416005 to 135416405 of the chromosome 5 are shown in SEQ ID NO: 1.

More preferably, measuring the methylation level of the CpG region of the mir-886 gene in the present invention may mean measuring the methylation level of cytosine located at position 135416205 (209th position in SEQ ID NO: 1) of chromosome 5 .

Also, preferably, measuring the methylation level of the CpG region of the INS-IGF2 gene in the present invention may mean measuring the methylation level of cytosine at the CpG site present in the 2161281 to 2161681 base of chromosome 11. In the present invention, the 2161281 to 2161681 base of the chromosome 11 is represented by SEQ ID NO: 2.

More preferably, measuring the methylation level of the CpG region of the INS-IGF2 gene in the present invention may mean measuring the methylation level of cytosine located at 2161481 (200 th position in SEQ ID NO: 2) of chromosome 11.

In addition, abnormal methylation changes of maternal genes show considerable similarity to methylation changes of genomic DNA obtained from biological samples such as cells, whole blood, serum, plasma, saliva, sputum or urine. Therefore, when the marker of the present invention is used For diagnosis of prematurity or prediction of risk of premature birth, it is possible to diagnose easily through blood or body fluids.

In the present invention, the agent for measuring the methylation level of the CpG region includes a compound that modifies an unmethylated cytosine base or a methylation-sensitive restriction enzyme, a primer specific to the methylated allele sequence of the gene, and a primer specific to the unmethylated allele sequence Gt; primer. ≪ / RTI >

The compound capable of modifying the unmethylated cytosine base may be bisulfite or a salt thereof, but is not limited thereto, preferably sodium bisulfite. Methods for detecting the methylation of a CpG site by modifying an unmethylated cytosine residue using such a bisulfite are well known in the art (WO01 / 26536; US2003 / 0148326A1).

In addition, the methylation sensitive restriction enzyme may be a restriction enzyme capable of specifically detecting the methylation of the CpG site, and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme. For example, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI, and the like. The methylation or non-methylation of the restriction enzyme recognition site at C may or may not be cleaved by restriction enzymes and may be detected by PCR or Southern blot analysis. Other methylation sensitive restriction enzymes than the above restriction enzymes are well known in the art.

As a representative method for measuring the level of methylation at the CpG site of a maternal mir-886 and / or INS-IGF2 gene, genomic DNA is obtained from a biological sample of a patient and transformed into DNA obtained by modifying a non- methylated cytosine base A compound or a methylation-sensitive restriction enzyme, amplifying the treated DNA by PCR using a primer, and confirming presence or absence of the amplified product.

Thus, the formulations of the invention may include primers specific for the methylated allele sequence of the mir-886 and / or INS-IGF2 gene and primers specific for the unmethylated allele sequence. In the present invention, the term "primer" means a short nucleic acid sequence capable of forming a base pair with a complementary template with a nucleic acid sequence having a short free 3-terminal hydroxyl group and serving as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In addition, primers can incorporate additional features that do not alter the primer properties of primers that serve as a starting point for DNA synthesis, as sense and antisense nucleic acids with 7 to 50 nucleotide sequences.

The primer of the present invention can be preferably designed according to the sequence of a specific CpG site to be subjected to methylation analysis and includes a pair of primers capable of specifically amplifying cytosine that has been methylated and not modified by bisulfite, And a primer pair that is not methylated and can specifically amplify cytosine modified by bisulfite.

In addition to the above preparation, the composition and kit may further contain a polymerase agarose, a buffer solution necessary for electrophoresis, and the like.

In another aspect, the present invention relates to a method for diagnosing the risk of premature birth by measuring the level of methylation at the CpG site of one or more genes selected from the group consisting of mir-886 and INS-IGF2.

In another aspect, the present invention relates to a method for measuring the methylation level at the CpG site of at least one gene selected from the group consisting of mir-886 and INS-IGF2 to provide information necessary for diagnosis of premature rash.

For example, the present invention relates to a method for detecting methylation level of a CpG region of at least one gene selected from the group consisting of mir-886 and INS-IGF2 from a maternal blood sample; And

Comparing the methylation level to the methylation level of the CpG region of the gene of the control sample.

And to provide information necessary to diagnose premature rupture.

Preferably, the control sample may be a sample of normal delivery mother.

In addition, the above method can also be applied to the case where the methylation level of the CpG region of the mir-886 gene measured in the maternal sample is higher than the methylation level measured in the control sample, or the CpG region methylation level of the INS-IGF2 gene measured in the maternal sample, If the level of methylation is lower than the measured level in the sample, it may further comprise determining that the mother is at risk for premature birth.

The term "biological sample" in the present invention refers to a sample such as a tissue, cell, whole blood, serum, plasma, saliva, sputum or urine which have different methylation levels of mir-886 and / or INS-IGF2 gene between premature- But are not limited thereto. Preferably a blood or body fluid sample.

First, to obtain genomic DNA from a mother and measure the methylation level, the genomic DNA can be obtained by the phenol / chloroform extraction method, SDS extraction method (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), Cetyl Trimethyl Ammonium Bromide (Murray et al., Nuc. Res., 4321-4325, 1980) or commercially available DNA extraction kits.

The step of measuring the methylation level of the CpG region of the gene may comprise detecting a compound or methylation sensitive restriction enzyme that modifies the unmethylated cytosine base, a primer specific to the methylated sequence of the gene CpG region, and a primer specific to the unmethylated sequence . ≪ / RTI >

More specifically, the step of treating the genomic DNA in the obtained sample with a compound or methylation-sensitive restriction enzyme that transforms the unmethylated cytosine base; And

The DNA thus processed is subjected to methylation-specific polymerase chain reaction and real-time methylation-specific polymerase chain reaction (PCR) using a primer capable of amplifying the methylation site of the gene CpG region reaction, PCR using methylated DNA-specific binding protein, quantitative PCR, pyrosequencing, and bisulfite sequencing, and measuring the methylation level.

In the above, the compound that transforms the unmethylated cytosine base may be bisulfite, preferably sodium bisulfite. Methods for detecting the methylation of a gene by modifying an unmethylated cytosine residue using such a bisulfite are well known in the art.

In addition, as described above, the methylation-sensitive restriction enzyme may be a restriction enzyme capable of specifically detecting methylation of a specific CpG site and may be a restriction enzyme containing CG as a recognition site of a restriction enzyme, for example, SmaI SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI, and the like.

As described above, the primers can be desirably designed according to the sequence of the specific CpG site to be subjected to the methylation analysis, and can be specifically designed to amplify cytosine that has been methylated and not modified by bisulfite Primer pair and a pair of primers that can specifically amplify cytosine that is not methylated and modified by bisulfite.

The measurement of the methylation level can be performed according to a method known in the art, for example, by performing electrophoresis to detect a band at a desired position. For example, when a compound that modifies an unmethylated cytosine residue is used, two kinds of primer pairs, that is, a pair of primers capable of specifically amplifying cytosine that has been methylated and not modified by bisulfite, The degree of methylation can be determined according to the presence or absence of the PCR product amplified by the primer pair capable of specifically amplifying cytosine modified by the Fight. Preferably, the methylation can be determined by treating the sample genomic DNA with bisulfite, amplifying the CpG region of the gene by PCR, and analyzing the base sequence of the amplified region using a bisulfite genomic sequencing method .

In the case where restriction enzymes are used, it is judged that the gene is methylated when there is PCR result in the DNA treated with the restriction enzyme in the state that the PCR product is displayed in the mock DNA, If there is no PCR result in the DNA treated with the enzyme, it can be judged whether or not the gene is methylated by judging that the gene is unmethylated, which is obvious to a person skilled in the art. In the above, mock DNA refers to a sample DNA that has been separated from the sample and has not undergone any treatment.

According to this method, when the CpG region of the mir-886 gene in the maternal sample is specifically hypermethylated or the CpG region of the INS-IGF2 gene is specifically measured in a low methylation state, it is predicted that there is a risk of premature birth You can.

Therefore, according to the present invention, it is possible to easily determine the risk of premature birth by effectively checking whether CpG is methylated in the mir-886 and / or INS-IGF2 gene.

The present invention can be used to early diagnose the risk of premature birth, prevent premature birth, and prevent serious complications and sequelae of newborn infants such as neonatal morbidity and cerebral palsy.

Figure 1 shows the results of quantitative analysis of the methylation level of mir-886 in the blood of premature maternal (left) and normal (right) mothers through pyrosequencing. The vertical axis indicates the degree of methylation (%), and the symbol "* " indicates p < 0.05.
FIG. 2 shows the results of quantitative analysis of the methylation level of INS-IGF2 in the blood of premature mothers (left) and normal mothers (right) through pyrosequencing. The vertical axis indicates the degree of methylation (%), and the symbol "* " indicates p < 0.05.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Research subjects

From June to December 2014, maternal admission to Ewha Womans Univ. Mokdong Hospital (Korea) was confirmed by maternal age of 37 weeks or less (n = 5) (N = 5). Maternal blood samples were collected and screened for methylation. In addition, methylation screening showed that there was a significant difference in methylation in the blood between the premature group and the normal group. In the screened gene, 41 patients with less than 37 weeks of preterm delivery and 40 patients with normal delivery after 37 weeks had additional blood And the methylation level difference was verified using pyrosequencing.

Example  2. genomic DNA  Extraction of

Genomic DNA was extracted from the collected maternal blood using Qiagen mini kit (Qiagen). The extraction method was carried out according to the manufacturer's manual. The extracted genomic DNA was quantitated by spectrophotometer and the DNA status was confirmed by electrophoresis on 1% agarose gel.

Example  3. Bisulfite ( Bisulfite ) process

When bisulfite is treated with genomic DNA, cytosine at the 5'-CpG-3 'site in the DNA sequence is retained as it is, but when it is unmethylated, it is converted to uracil to measure the degree of methylation . Therefore, genomic DNA was treated with bisulfite to distinguish methylated cytosine from unmethylated cytosine. First, ~ 700 ng of genomic DNA was treated with Zymo EZ DNA Methylation Kit (Zymoresearch Inc.) according to the manufacturer's manual. The bisulfite-treated DNA thus prepared was dissolved in H ₂ O and stored at -20 ° C Respectively.

Example  4. DNA Methylation Microarray

A DNA methylation microarray was performed using the Infinium (®) Human Methylation 450K BeadChip. Following the manufacturer's instructions, the bisulfite-treated DNA was amplified, fragmented, precipitated and resuspensioned and hybridized to the BeadChip. After washing, BeadChip was scanned using HiScan SQ system (Illumina) and β value was calculated using Genome Studio software (Illumina). The degree of DNA methylation is expressed as a value having a value of 0 to 1, and a value of 0 means that the corresponding CpG site is completely unmethylated and 1 means completely methylated.

Example  5. Pregnancy vs. Pregnancy Haploid  Quantitative methylation validation

To amplify the methylation gene of the candidate gene, each designed primer was used to amplify the bisulfite pretreated DNA.

miR886 Forward primer: GtAAAGTTAAAAGGGATAAAAAA (SEQ ID NO: 3)

miR886 Reverse primer: AtAGAGATGGAtAGATAGAAAGTt (SEQ ID NO: 4)

INS-IGF2 Forward primer: TtTGGGttAGGtTTGGAGtt (SEQ ID NO: 5)

INS-IGF2 Reverse primer: GTtTGGGGAAAttATtTttTGG (SEQ ID NO: 6)

PCR conditions were amplified using Surecycler-8800 (Agilent) at 95 ° C for 10 minutes, 45 cycles (95 ° C for 30 seconds, 48 ° C for 30 seconds, 72 ° C for 30 seconds) and 72 ° C for 5 minutes. For bi-pyrosequencing analysis, biotin-conjugated amplification products were coupled to beads with binding buffer with streptavidin-separase bead. Streptavidin-Sepharose beads conjugated with the amplified product were adsorbed on a PSQ 96 sample polypropylcell containing a 96 magnetic ejectable microcylinder, denatured and washed. Beads were added to the annealing buffer using the primers for analyzing the base [miR886: ATAAAAGGGTtAGTAAGtAt (SEQ ID NO: 7), INS-IGF2: GTTGGAtTTGTATAGA (SEQ ID NO: 8)] and denatured at 80 ° C for 2 minutes. At the room temperature, the amplification products were complementarily bound to the starting analyte, and quantitative methylation of the estrogen - related genes was verified using PyroMark ID (Qiagen).

Experiment result

Among the total 485,577 CpG sites, 10 gene promoters showed hypermethylation more than 2 times higher than normal in the maternal blood of pregnant women and normal pregnant women. , And thirteen gene promoters showed hypomethylation more than twofold. Among them, mir-886, which showed hypermethylation and INS-IGF2, which showed low methylation, compared to normal mothers were selected (p <0.05, Table 1).

Genes showing significant methylation differences of more than 2-fold in normal and maternal blood gene Gene name Genebank registration number chromosome CpG site Hypermethylation in preterm birth) mir-886 Homo sapiens vault RNA 2-1 (VTRNA2-1), vault RNA NR_030583 5 chr5: 135416005
-135416405 Hypomethylation in preterm birth at the risk of preterm birth INS-IGF2 INS-IGF2 readthrough NR_003512 11 chr11: 2161281
-2161681

Table 2 shows the results of calculating the beta value through a DNA methylation microarray. The degree of DNA methylation is expressed as a value having a value of 0 to 1, and a value of 0 means that the corresponding CpG site is completely unmethylated and 1 means completely methylated. mir-886 was hypermethylated in maternal maternal group and INS-IGF2 in maternal maternal group.

probe ID Gene beta value normal
Mother group Maternity maternity cg04481923 mir-886 0.1217 0.3892 * cg04072545 INS-IGF2 0.1421 0.0657 *

        * p < 0.05

Table 3 shows the results of further blood sampling from 41 preterm and 40 normal pregnancies for mir-886 and INS-IGF2 genes, which showed significant methylation changes in preterm and maternal groups, using pyrosequencing The difference in methylation level is verified. The results are shown as an average after quantitative analysis, and the graphs are shown in FIGS. 1 and 2.

(unit: %) Maternity maternity
(n = 41) Normal mother group
(n = 40) P value mir-886 35.55 ± 3.17 27.43 + - 3.53 0.034 * INS-IGF2 3.23 ± 0.42 4.51 + - 0.38 0.013 *

* p < 0.05

<110> Ewha University - Industry Collaboration Foundation <120> Composition for predicting a risk of preterm delivery using CpG          methylation status of gene and uses thereof <130> DPP20152964KR <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 400 <212> DNA <213> Homo sapiens <220> <221> gene &Lt; 222 > (1) .. (400) <223> chr5: 135416005-135416405 <400> 1 tacctggcag tacaggctgg tcacgcacgc cctgtaagac cagtggccag cctccatact 60 ttctgtcaca ccttcaaagt gacaccaact tatgttatca gcctattaat ttagcaaagt 120 taaaagggat aaaaaataac aaagccttca gagtgacaga ttataccgaa tttattgcat 180 aaaagggtca gtaagcaccc gcgggtctcg aaccccagca cagagatgga cagatagaaa 240 gtccggcatg aggaggtaac cgcttgagct aactccgacc cgggtaggag tgtgcgactg 300 aaacttctaa accatagaag agtgacgatg tggagaggga cgggctgcat gtgctccccg 360 cccccgagag gcctgcgtca tgcggtctcg cccgctctgc 400 <210> 2 <211> 400 <212> DNA <213> Homo sapiens <220> <221> gene &Lt; 222 > (1) .. (400) <223> chr11: 2161281-2161681 <400> 2 atctgggcca ggcttggagc ctacggaggc tccggccgcc agaggagaga ggatcagggc 60 gggaaacaca gctcaaatcc tacctgcatg gccgagctca ccggggtgcg tctaagtagc 120 tcgcctttgc ggcccaccca aaatatctgg ataatggtta ccccgtcctc agtgcgttgg 180 acttgcatag acgcgagttc ggtctcgggg gccaccacga taatttgggg gtctggggaa 240 accatctcct ggagagtttg aacgatgtaa gaaagcactg tgatttttac caagtcaaaa 300 accacgcaca aaatcccgca ccccgccagc cccccgccgc ctaagcctag ccaaggggaa 360 ggggccgccg gcggaatctc gcgcccccct ggcccccctc 400 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> miR886 Forward primer <400> 3 gtaaagttaa aagggataaa aaa 23 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> miR886 Reverse primer <400> 4 atagagatgg atagatagaa agtt 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> INS-IGF2 Forward primer <400> 5 tttgggttag gtttggagtt 20 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> INS-IGF2 Reverse primer <400> 6 gtttggggaa attatttttt gg 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miR886 primer <400> 7 ataaaagggt tagtaagtat 20 <210> 8 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> INS-IGF2 primer <400> 8 gttggatttg tataga 16

Claims (13)

a methylation level of the CpG region of one or more maternal genes selected from the group consisting of mir-886 (microRNA-886) and INS-IGF2 (INS-IGF2 readthrough)
Compositions for diagnosing premature rupture.
2. The method according to claim 1, wherein the agent for measuring the methylation level of the CpG region of the gene comprises
Compounds or methylation sensitive restriction enzymes that modify unmethylated cytosine bases;
A primer specific for the methylated sequence of the CpG region of the gene; And
Wherein the primer comprises a primer specific for the unmethylated sequence.
3. The composition of claim 2, wherein the compound that modifies the unmethylated cytosine base is bisulfite or a salt thereof.
3. The composition of claim 2, wherein the methylation sensitive restriction enzyme is Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI or Not I.
The composition according to claim 1, wherein the CpG site of the mir-886 gene comprises CpG which appears in the nucleotide sequence of SEQ ID NO: 1 (135416005 to 135416405 th of chromosome 5).
2. The composition according to claim 1, wherein the CpG site of the INS-IGF2 gene comprises CpG which appears in the nucleotide sequence of SEQ ID NO: 2 (2161281 to 2161681 th of chromosome 11).
7. A composition comprising a composition according to any one of claims 1 to 6,
Pregnancy risk diagnostic kit.
Measuring the methylation level of a CpG site of at least one gene selected from the group consisting of mir-886 and INS-IGF2 from a maternal sample; And
Comparing said methylation level with a methylation level of a CpG region of a gene of a normal delivery mother;
How to provide information necessary to diagnose premature rupture.
9. The method according to claim 8, wherein the step of measuring the methylation level of the CpG region of the gene comprises:
Treating the genomic DNA in the obtained sample with a compound or methylation-sensitive restriction enzyme that modifies the unmethylated cytosine base; And
The DNA thus processed is subjected to methylation-specific polymerase chain reaction and real-time methylation-specific polymerase chain reaction (PCR) using a primer capable of amplifying the methylation site of the gene CpG region reaction, PCR using methylated DNA-specific binding protein, quantitative PCR, pyrosequencing, and bisulfite sequencing to determine the methylation level.
10. The method of claim 9, wherein the compound that modifies the unmethylated cytosine base is bisulfite or a salt thereof.
10. The method of claim 9, wherein the methylation sensitive restriction enzyme is SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI or NotI.
9. The method according to claim 8, wherein the CpG site of the mir-886 gene comprises CpG which appears in the nucleotide sequence of SEQ ID NO: 1 (135416005 to 135416405 th of chromosome 5).
9. The method according to claim 8, wherein the CpG site of the INS-IGF2 gene comprises CpG which appears in the nucleotide sequence of SEQ ID NO: 2 (2161281 to 2161681 th of chromosome 11).

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