KR20150032062A - Fetal Epigenetic Markers and Use thereof - Google Patents

Abstract

The present invention relates to a fetal specific epigenetic marker and a use thereof. The present invention provides a specific epigenetic marker X1, X2, and X3, and a method for a non-invasive prenatal diagnosis and the detection of a fetal DNA from a blood of a pregnant woman using the same. A composition for the detection of the fetal DNA according to the embodiment of the present invention includes one or more markers selected from a group composed of the fetal specific epigenetic marker X1 (sequence number 1), X2 (sequence number 2), and X3 (sequence number 3).

Description

Fetal Epigenetic Markers and Use thereof < RTI ID = 0.0 >

The present invention relates to fetal hemostatic markers and uses thereof.

The fetal DNA present in the blood of pregnant women is originated from the fetal placenta and is known to be released into the blood of pregnant women due to apoptosis through spontaneous apoptosis of the trophoblast during pregnancy (Prenat Diagn 2007; 27: 415-8, Am J Pathol 2006; 169: 400-4).

The fact that fetal DNA is present in the blood of pregnant women has been reported for the first time by detecting the Y chromosome specific DNA in the blood of pregnant women who have conceived a male fetus (Lancet 1997; 350: 485-487) And a new source of noninvasive prenatal diagnosis of fetal genetic disease.

The fetal DNA present in the blood of pregnant women is known to be increased in pregnancy related diseases due to abnormal placental conditions such as preeclampsia and fetal growth disorder (Am J Pathol 2006; 169: 400-4; Prenat Diagn 2007; 27: 415 Genes Dev 2000; 14: 3191-203; Am J Obstet Gynecol 2009; 200: 98.e1-6; Human Reproduction 2003; 18 (8): 1733-736) (Am J Obstet Gynecol 2002 Nov; 187 (5): 1217-21. Clin Chem 2003; 49: 239-242. Clin Chem 1999; 45: 1747-51).

Detection of fetal DNA present in the blood of existing pregnant women was carried out using a DNA sequence specific to the Y - derived Y chromosome which does not exist in maternal women. However, in this case, the fetus has a limitation that can not be applied to a girl. Therefore, in order to more accurately measure the fetal DNA present in the blood of pregnant women regardless of fetal sex, the epigenetic characteristics of genes such as DNA methylation, which shows a specific pattern depending on the stage and type of tissue, have.

Recently, research is underway to find fetal DNA identifiers using epigenetic differences between pregnant women's DNA and fetal DNA. For example, PDE9A, SERPINB5, RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2 and PYCARD are present irrespective of fetal sex and the level of methylation in pregnant blood and placenta is different. Is used to determine whether circulating fetal DNA is present. PDE9A and SERPINB5 have been reported to be mainly methylated in the blood cells of pregnant women and present in the unmethylated state in the placenta (Biol Reprod. 82 (4): 745-50, 2010). On the other hand, it is known that RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2, and PYCARD are present in the unmethylated state of the blood cells of pregnant women and in the methylation state of the placenta (US Patent No. 7754428).

The main objective of the current prenatal screening is to detect Down syndrome, a chromosomal disorder that occurs when three chromosomes are present in more than one chromosome. Down syndrome is an abnormality in the whole body including mental retardation, somatic anomaly, systemic dysfunction, and growth disorder.

At present, the diagnosis of Down syndrome is based on the combination of nuchal translucency ultrasound scan of the fetus and serum marker (pregnancy-associated plasma protein A, human chorionic gonadotrophin, alpha fetoprotein, unconjugated estriol, and inhibin-A) The test is in parallel. However, the accuracy of detection by this method has been reported to be approximately 84% at a false positive rate of 5% (Obstet Gynecol. 2006; 107: 167-173, N Engl J Med. 2005; 353: 2001-11). In general, Down's syndrome is diagnosed by chromosome analysis through amniocentesis or chorionic villus sampling. However, this amniocentesis or choriocarcinoma biopsy is an invasive method with a risk of approximately 1% abortion have. Therefore, non-invasive screening and diagnostic techniques with high accuracy for Down syndrome are still required for reducing the need for current invasive diagnostic methods and replacing screening methods.

A variety of fetal DNA identifiers have been reported that present a hematological difference between maternal DNA and fetal DNA present on chromosome 21 (Clin Chem 2008; 54: 500-11, Clin Chem 2010; 56: 90-8, Am J Pathol 2009 May; 174 (5): 1609-18). However, the reported fetal DNA identifiers were identified based on tissue-specific epigenetic characteristics between maternal blood cells and placental tissues for the detection of noninvasive fetal DNA, There is a limit to the use of the marker as a marker. Therefore, there is still a need for a marker that can be used more effectively for noninvasive prenatal diagnosis, including detection of fetal DNA in the blood of pregnant women.

The present inventors conducted a study to identify markers having histologic characteristics specific to fetal tissues that can be used for noninvasive prenatal diagnosis, and found a marker X1 of SEQ ID NO: 1, a marker X2 of SEQ ID NO: 2 , And marker X3 of SEQ ID NO: 3 distinguish fetal DNA from maternal DNA and can be used to distinguish fetuses with chromosomal abnormalities such as pregnancy-related diseases or Down's syndrome. Thus, the present invention has been completed.

It is an object of the present invention to develop a new fetal DNA-specific histologic marker showing tissue-specific proliferative characteristics present on chromosome 21 and to identify reliable fetal DNA-specific markers for fetal DNA detection in pregnant blood, To provide a novel DNA marker that is effective in detecting Down's syndrome as well as pregnancy related diseases caused by abnormal placental conditions.

It is still another object of the present invention to provide a method for detecting the presence and level of fetal DNA in non-invasive pregnant blood using a fetal specific hysteretic marker and obtaining information necessary for prenatal diagnosis.

One aspect of the invention is a composition for detecting fetal DNA, comprising at least one marker selected from the group consisting of fetal specific proximal markers X1 (SEQ ID NO: 1), X2 (SEQ ID NO: 2), and X3 .

It is known that fetal DNA and maternal DNA can differ in their methylation status and can be distinguished therefrom. Since methylation of DNA is a postmortem phenomenon and fetal specific prognostic markers X1, X2, and X3 have different methylation status compared to maternal DNA, the fetal DNA can be distinguished from maternal DNA by its methylation status. Fetal specific haplotype markers X1, X2, and X3 are present on chromosome 21 and are completely methylated in pregnant DNA, but are completely methylated or hypermethylated in fetal DNA and copy number variant (CNV) .

As used herein, the term "fetal specific haematological marker" refers to a specific region on a chromosome that can be used to specifically distinguish fetal DNA from maternal DNA by virtue of metastatic characteristics such as methylation. Fetal-specific hematologic markers may be useful in noninvasive prenatal diagnosis. By detecting the fetal DNA in the blood of the pregnant woman using the fetal specific hysteretic marker and judging the existence of the fetal DNA, it is possible to measure the level of the fetal DNA by detecting the fetal chromosomal abnormality, preeclampsia, Related diseases can be diagnosed.

In the present invention, the fetal-specific proliferative marker X1 is a region (GeneBank accession no. NG_029732; UCSC Genome Browser Assembly: GRCh37 / hg19) on the chromosome 21 from 39865787 to 39865951 and is represented by SEQ ID NO: 1.

In the present invention, the fetal specific proximal marker X2 is a region (GeneBank accession number AP001745; UCSC Genome Browser Assembly: GRCh37 / hg19) on the chromosome 21, and is represented by SEQ ID NO: 2.

In the present invention, the fetal specific proximal marker X3 is a region (GeneBank accession No. AP001745; UCSC Genome Browser Assembly: GRCh37 / hg19) on the chromosome 21 and represented by SEQ ID NO: 3.

The CpG site of the fetal specific proximal markers X1, X2, and X3 is completely methylated or hypermethylated in the fetal DNA and is completely in the unmethylated state in the maternal DNA, so DNA from the fetal and maternal species can be distinguished by methylation And quantitative evaluation of methylated DNA can be used to diagnose maternal anomalies. In addition, these markers can be used as positive controls for the presence of fetal DNA in noninvasive prenatal diagnosis.

In one embodiment of the invention, the marker can be used to detect the presence of fetal DNA in a polymerase chain reaction involving real-time PCR.

In one embodiment of the present invention, the step of physically separating the methylated DNA from the unmethylated DNA without chemical modification may be performed prior to performing the polymerase chain reaction to detect the presence of fetal DNA.

Physically separating the methylated DNA from the unmethylated DNA means separating the DNA itself by methylation, without chemically modifying or truncating it. Physical separation of methylated DNA can be achieved by hybridization using a microarray comprising a probe recognizing the methylation site or immunoprecipitation using a methylation-specific antibody. For example, a methyl-CpG binding domain capture (MBD) microarray can be used.

In one embodiment of the invention, the composition comprises a primer pair consisting of SEQ ID NOS: 4 and 5 for amplification of marker X1, a probe of SEQ ID NO: 6 for detection of marker X1, SEQ ID NOs: 7 and 8 for amplification of marker X2 , A probe of SEQ ID NO: 9 for the detection of the marker X2, and a probe of SEQ ID NO: 12 for the detection of the marker X3, and a primer pair of SEQ ID NOs: 10 and 11 for amplification of the marker X3 and a probe of SEQ ID NO: A primer pair and a probe.

In one embodiment of the invention, the composition further comprises a buffer, a dNTP mixture, and a DNA polymerase for performing a real-time PCR.

Real-time polymerase chain reaction simultaneously amplifies and measures the target nucleic acid. Since the intensity of the signal generated from the amplification product by the polymerase chain reaction, for example, fluorescence intensity is measured in real time, the result can be confirmed faster and more sensitively than the conventional polymerase chain reaction. In addition, there is no need for additional steps such as electrophoresis or sequencing for the analysis of the amplification product.

The term "probe" as used herein refers to a single-stranded nucleic acid molecule comprising a sequence complementary to a target sequence. Probes can be modified to the extent that the hybridization specificity is not impaired. For example, a reporter fluorescent substance or a quencher may be attached to the end of the probe.

In one embodiment of the present invention, a probe for use in a real-time PCR is attached with a fluorescent substance at the 5 'end and a small mineral substance at the 3' end.

In one embodiment of the invention, the fluorescent material is selected from the group consisting of 6-Carboxyfluorescein, TET (2 ', 7'-dichloro-6-carboxy-4,7-dichlorofluorescein), 6-JOE 4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein), HEX (2 ', 4', 5 ', 7'- tetrachloro-6-carboxy-4,7-dichlorofluorescein), Cy3 (Cyanine 3) Cy5, and VIC. However, the present invention is not limited thereto.

In one embodiment of the present invention, the minerals are selected from the group consisting of 6-carboxytetramethylrhodamine, BHQ (Black Hole Quencher) -1, BHQ-2, BH3-3 and ROX (carboxy-X- But is not limited thereto.

In one embodiment of the present invention, a minor groove binder (MGB) may be attached to the 3'-end of the probe. MGB contributes to the accuracy of the analysis by increasing the stability of the double strand formed.

In one embodiment of the invention, the single or multiple real-time PCR reactions can be performed under conventional conditions known in the art or can be readily determined by one of ordinary skill in the art, The fluorescence from the probe with the fluorescent material is measured in the middle annealing and extension steps.

Another aspect of the present invention is a method for detecting fetal DNA in pregnant blood,

Physically separating the methylated DNA from the maternal blood sample,

Detecting the presence or absence of one or more of the fetal specific haplotype markers X1 (SEQ ID NO: 1), X2 (SEQ ID NO: 2), and X3 (SEQ ID NO: 3) for the separated methylated DNA .

Fetal specific haplotype markers X1, X2, and X3 are fully methylated or hypermethylated in fetal DNA, that is, placenta-derived DNA, and unmethylated in maternal DNA. Therefore, the presence of fetal DNA can be confirmed by physically separating the methylated DNA from the unmethylated DNA in the pregnant blood sample and detecting the markers X1, X2, X3, or a combination thereof from the unmethylated DNA. It is also possible to provide information necessary for prenatal diagnosis by measuring the level of the markers X1, X2, X3, or a combination thereof and comparing it with a standardized standard.

In one embodiment of the invention, the step of physically separating the methylated DNA from the pregnant blood is carried out by methylated DNA-specific immunoprecipitation or methylated DNA-specific microarrays.

The step of physically separating the methylated DNA from the unmethylated DNA can be accomplished by binding the methylated DNA to the antibody using a methylated DNA specific antibody and then releasing the methylated DNA from the precipitated antibody complex or by using the methylated CpG binding domain protein Followed by hybridization to a microarray containing the probe, followed by releasing the hybridized DNA. The methylated DNA is isolated from physically unmethylated DNA without chemically modifying or truncating it, from which the presence of a fetal specific haploid marker is detected or its level measured.

In one embodiment of the present invention, the step of detecting the presence or absence of one or more of said fetal-specific hemostatic markers X1, X2, and X3 may comprise performing a real-time PCR.

In one embodiment of the present invention, the real-time PCR reaction comprises a primer pair consisting of SEQ ID NOS: 4 and 5 for amplification of marker X1, a probe of SEQ ID NO: 6 for detection of marker X1, a sequence for amplification of marker X2 A pair of primers consisting of SEQ ID NOs: 10 and 11 for amplification of marker X3, and a pair of primers of SEQ ID NO: 12 for the detection of marker X3. , And a probe.

In one embodiment of the invention, the method for detecting fetal DNA in pregnant blood according to the present invention comprises the steps of measuring the level of at least one of the fetal specific prognostic markers X1, X2, and X3 on the isolated methylated DNA, And

And comparing the level of the measured marker to a standardized reference for the fetal specific prognostic marker.

The level of the measured marker relative to a standardized standard for the fetal specific hysteretic marker provides information necessary for diagnosis of a pregnancy related disorder or a Down syndrome fetus.

The level of the marker in the sample means the amount of the marker present in the sample, which can be expressed in various quantitative units such as the number of copies of the corresponding marker in the sample.

The standardized criteria for the fetal specific prognostic marker may be the level of the marker measured from the blood of a normal fetus in a pregnant woman or the level of a marker measured from the blood of a pregnant woman with a Down syndrome fetus.

If the level of the fetal specific hysteretic marker measured from the sample is higher than the level of the marker measured from the blood of a normal pregnant woman or similar to the level of the marker measured from the blood of a pregnant woman The fetal DNA of the sample can be judged to be derived from the Down syndrome fetus.

In addition, the amount of fetal DNA in pregnant blood is known to increase in various pregnancy related diseases such as preeclampsia and fetal growth and development disorder. Thus, by measuring the level of fetal-specific haploid marker and comparing it with standardized criteria, it is possible to provide information necessary for diagnosing pregnancy related diseases.

The standardized criteria for the fetal specific hysteretic marker may be the level of the marker measured from a normal pregnant woman or the level of a marker measured from a pregnant woman with a pregnancy related disorder.

If the level of fetal-specific haploid marker measured from the sample is higher than the level of the marker measured from the blood of a normal pregnant woman or similar to the level of a marker measured from the blood of a pregnant woman with a pregnancy-related disorder, The fetal DNA can be judged to be derived from placental abnormalities due to pregnancy related diseases.

The term "standardized reference" used in connection with the level of a marker herein refers to a value for comparing with the level or amount of a marker in a sample and may be an average value obtained from a group that can be a reference of the sample to be measured . For example, a standardized criterion for determining whether a person has Down's syndrome may be an average of values measured for pregnant women with normal fetuses, or an average of values measured from pregnant women who have pregnant women with Down's syndrome, For diagnosing a pregnancy related disease, it may be an average of the values measured from healthy pregnant women of the same or similar pregnancy without having a pregnancy related disease, or an average of the values measured from pregnant women with a pregnancy related disease.

As used herein, the term " pregnancy related disorder "means a condition or disease that may affect a pregnant woman, a fetus of a pregnant woman, or both. Such conditions or illnesses can manifest itself during pregnancy or delivery. Examples of pregnancy related diseases include, but are not limited to, ectopic pregnancy, preeclampsia, and premature labor.

In one embodiment of the invention, the step of detecting the presence or absence of one or more of said fetal specific posterior markers X1, X2, and X3 is performed on a combination of fetal specific posterior markers X1, X2, and X3 . The presence or absence of the fetal DNA can be detected by detecting each marker or a combination thereof.

 Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

The composition and method according to one embodiment of the present invention can be used to noninvasively detect the presence of fetal DNA in a maternal blood sample and obtain information necessary for prenatal diagnosis such as Down's syndrome and pregnancy related diseases.

Figures 1 to 3 show non-sphinx sequencing data for fetal specific proliferative markers X1 (four CpG sites), X2 (seven CpG sites), and X3 (eight CpG sites), respectively. All of the selected markers showed hypermethylation or complete methylation in fetal placental tissue, but complete methylation in maternal peripheral blood.
4 in Fig. 4 shows the results of qPCR for the fetal specific prophylactic markers X1 (SEQ ID NO: 1), X2 (SEQ ID NO: 2), and X3 (SEQ ID NO: 3), respectively, performed on the maternal blood sample.
Figure 7 shows the multi-real-time qPCR results of the fetal specific posterior marker X2 and the reference marker RASSF1A.
Figure 8 shows the analysis of the ROC curve obtained from the multi-real-time qPCR of the fetal-specific haplotype marker X2 and the reference marker RASSF1A. In the figure, UMO denotes marker X2, and RATIO denotes a ratio index calculated as a ratio of blood concentration of marker X2 / blood concentration of RASSF1A.

Example  1. Fetal-Specific Histology Marker  Screening

The chromosome 21 was screened for fetal-specific hematological markers that could be used to detect fetal DNA in maternal blood. Markers with different methylation patterns in pregnant women and fetuses were selected and confirmed to be applicable as fetal - specific prognostic markers.

(1) Target

We recruited pregnant and non-pregnant women with normal fetus or Down syndrome (T21) fetuses from March to December 2011 at Cheil Hospital Obstetrics and Gynecology. We obtained the permission (# CGH-IRB-2011-85) for the study from the Jeil Hospital Ethics Committee. All patients agreed in writing about sample collection and follow-up studies.

(2) Sample processing

Methyl-CpG Binding Domain Protein Capture (MBD) - For microarray analysis, maternal peripheral blood samples were collected in a tube containing EDTA immediately prior to chorionic villus sampling in the first trimester. All placenta samples were obtained from chorionic villus sampling. Peripheral blood samples were centrifuged at 1,600 g for 10 minutes, and the obtained peripheral blood cell portions were re-centrifuged at 2,500 g for 10 minutes, and the residual plasma was removed to obtain peripheral blood cells. DNA was extracted from peripheral blood cells and placental tissues using QIAamp Blood Kit (Qiagen) and QIAamp Tissue Kit (Qiagen), respectively.

(3) Array Hybridization

For tiling oligonucleotide arrays of chromosome 21, whole blood samples from normal non-pregnant women (n = 2), whole blood samples from women with normal pregnancy (n = 4) and fetal placenta samples n = 4), and fetal placenta samples from normal women (n = 4) who were pregnant with Down syndrome fetuses.

After sonication of each DNA sample from placenta tissue and blood cells, only the methylated DNA was selectively bound using MBD that specifically recognizes the methylated CpG dinucleotide, and the bound MBD-DNA Only the methylated DNA was selectively extracted using magnetic beads that can be specifically bound (Invitrogen, MethylMiner ^TM Methylated DNA Enrichment Kit). The products obtained through MBD were amplified using a Whole Genome Amplification Kit (Sigma) capable of amplifying the whole genome, and each amplified DNA was labeled with Genomic DNA Enzymatic Labeling Kit (Agilent), and chromosome 21 ≪ / RTI > to a specifically tailored chip for < RTI ID = 0.0 > The array platform was designed for a high-resolution tiling oligonucleotide array specific for chromosome 21 (Agilent technologies, Design_ID038569). The array consists of 400,000 50-mer or 60-mer oligonucleotides covering the entire chromosome 21 with one median probe density per 60 bp. A microarray protocol, including labeling, hybridization, and post-hybridization procedures, has been proposed by the manufacturer and can be found at http://www.agilent.com.

In summary, the labeled MBE (Cyanine 5-dUTP) DNA and reference DNA (Cyanine 3-dUTP) were washed using Amicon filters (Millipore). Competitive hybridization to the microarray was then carried out using a Methylation Hybridization Kit (Agilent) at 67 ° C for 40 hours in a rotating SureHyb chamber according to the manufacturer's instructions. The washed slides were then scanned using a High-Resolution C Scanner (Agilent) and fluorescence was evaluated using Feature Extraction software (Agilent Technologies). Intensity lowess normalization was performed using the obtained fluorescence values and the corrected values were evaluated for methylation through BATMAN (Bayesian tool for methylation analysis) algorithm. From the values evaluated according to the BATMAN algorithm, values with a log value of 1 or more and a BATMAN call of 1 were evaluated as methylated.

Data analysis of tiling oligonucleotide arrays

Two signal values are normalized using background correction and signal ratio (MBD / input), signal log ratio [log ₂ (MBD / input)], P [X] And P were obtained. The log ₂ value is the Cy5: Cy3 fluorescence ratio (methylated DNA: total input DNA recovered by MBD) for each probe, converted to a log ₂ scale, and represents the relative measure of the amount of methylated DNA in each locus. The present inventors applied median and lowess normalization to raw data and filtered outlier probes to remove low-quality data points. The P [X] and P values used to obtain the binding call in the MBD array analysis are defined as the probability that the X value deviates from the Gaussian distribution of the X value of the entire genome of the sample. The X value for the probe means the difference between the MBD signal and the input signal corrected for the symmetry of the distribution. The value for the probe was calculated as the average X, taking into account the signal of the adjacent probe (probe within 1 kb of the probe). Finally, we define full methylation as normalized log ₂ ratio > 1 and convert complete methylation to normalized log ₂ Ratio = 0.

In the tiling oligonucleotide array assay, candidates for fetal specific prognostic markers were selected by identifying regions of differentially methylated regions in blood and placental tissues. Regardless of the presence or absence of disease, a total of three regions were selected as marker candidates based on the unmethylation defined in the placenta sample (n = 8) as a full methylation defined as a normalized log ₂ ratio greater than 1 and a normalized log2 ratio of 0 did. Table 1 below shows the results.

The areas identified by the probes designated Index 213672, 243799, and 243805, which exhibited complete methylation in fetal placenta samples and which were found to exhibit complete unmethylation in all blood samples, with or without pregnancy, were designated as fetal-specific markers X1, X2 and X3.

(4) Copy Number Variation in Target Area Confirm

The DNA sequences of the fetal specific prognostic markers X1, X2 and X3 were cloned by searching the database of genomic variants (DGV) (http://projects.tcag.ca/variation/) to determine the copy number variant (CNV) And the presence or absence thereof. The target area without CNV was used to verify its potential for non-invasive fetal markers through the detection of maternal plasma. Table 2 below shows the area finally confirmed to be free of CNV. The red sequence represents the probe sequence selected by the microarray and the underlined region is the probe sequence of the selected probe.

(5) Bisulfite Direct sequencing direct sequencing )

The accuracy of the array analysis data was confirmed using non-sphygite direct sequencing for selected markers by tiling oligonucleotide array analysis. A DNA sample (1 ㎍) was non-sulfated-transformed using EpiTect Bisulfite Kit (Qiagen) according to the manufacturer's instructions. The bisulfite-transformed DNA was amplified by PCR using a primer for distinguishing methylated CpG and unmethylated CpG sites. The PCR primers used were as follows:

Index ID: 213672: Forward primer: 5'-TTGTGTGTGGTATTTGTTTTG-3 '

Reverse primer: 5'-CACACTTACATTTACTTCCTTCC-3 '

Index ID: 243799: Forward primer: 5'-GATGCGTTAGATTTAAGGGAGG-3 '

Reverse primer: 5'-CTCACTCTCACGAAACCCCTC-3 '

Index ID: 243805: Forward primer: 5'-TGTTAGGATAGGAGTAGTGG-3 '

Reverse primer: 5'-CCCACAAACGCTAAACATCTC-3 '

The PCR reaction solution contained 10 ng genomic DNA, 10 pM primer, 0.25 mM dNTP, 1.5 mM MgCl ₂ , 1 X buffer, and 0.25 U Taq polymerase per 20 μl total reaction volume. The PCR conditions included pre-denaturation at 95 캜 for 10 min, 30 sec at 95 캜, 30 sec at 56 캜, 35 cycles consisting of 40 sec at 72 캜, and a final extension at 72 캜 for 10 min. After PCR amplification, the PCR product was purified using a PCR purification kit (Bioneer) according to the manufacturer's instructions and the sequence was determined using the PRISM BigDye Terminator Cycle Sequencing Kit (Applied Biosystems Inc.). The sequencing products were analyzed using a PRISM 3100 Genetic Analyzer (Applied Biosystems Inc.) and electrophorograms were recorded using Genescan software version 3.7 (Applied Biosystems, Inc.). Corresponding genotypes were placed using Genotyper software version 3.7 (Applied Biosystems, Inc.). Figures 1 to 3 show non-sphinx sequencing data for markers X1, X2, and X3, respectively. Selected fetal specific haematological markers X1, X2, and X3 were identified as SEQ ID NOS: 1-3, respectively. Markers X1, X2, and X3 all showed hypermethylation or complete methylation in fetal placental tissue, but complete methylation in maternal peripheral blood.

Example  2. Fetus DNA Detection

Detection of fetal DNA was performed on samples obtained from pregnant blood using the fetal specific hysteretic markers X1, X2, and X3 selected in Example 1.

(1) Isolation of cell-free DNA from maternal plasma

Peripheral blood from each pregnant participant (n = 6) was collected in EDTA tubes. Immediately after harvesting, peripheral blood samples of the maternal blood were centrifuged at 4 ° C to 1,600 g for 10 minutes. Plasma was resuspended at 16,000 g for 10 min to minimize further release of maternal DNA and stored at -80 ° C in a deep freezer until testing. According to the manufacturer's instructions, cff (cell free fetal) -DNA was extracted from 1 mL of maternal plasma using QIAamp DSP Virus Kit (Qiagen). Each DNA sample was eluted with 30 [mu] l of sterile DNase-free water and labeled for subsequent blinded analysis.

(2) Enrichment of methylated DNA

The methylated DNA was isolated from circulating DNA extracted from maternal plasma using a MethylMiner ^TM methylated DNA enrichment kit (Invitrogen). The MBD-biotin protein (3.5 μg) was coupled to 10 μl Dynabeads M-280 Streptavidin and the MBD-magnetic bead conjugate was then washed three times and resuspended in 1 volume of 1 × Bind / Wash buffer . 30 [mu] l of extracted circular DNA on a rotary mixer was added to MBD-magnetic beads and capture reaction was carried out at 4 [deg.] C for 24 hours. The beads were then washed three times with 1X Bind / Wash buffer and the bound methylated DNA was eluted by stepwise elution using increasing NaCl concentrations [450 mM, 600 mM, 1,000 mM, and 2,000 mM NaCl] in elution buffer Eluted. The eluted DNA was concentrated using a DNA concentrator (Zymo research corp.) And eluted with a final volume of 30 l. The methylation DNA was isolated using the control DNA (methylated DNA and unmethylated DNA) contained in the kit.

(3) Quantitative real-time polymerase chain reaction (q-RT PCR)

Q-RT PCR was performed to confirm the possibility of detection of fetal-specific hematopoietic markers in maternal plasma. q-RT PCR was performed using the ABI 7500 system (ABI). 10 [mu] l of 2X Master Mix II (Kogene biotech) and 5 [mu] l of methylated plasma DNA captured by MBD were used to prepare a reaction mixture in a volume of 20 [mu] l. Primers and hydrolysis probes were used at final concentrations of 200 nmol / L and 100 nmol / L, respectively. The sequences of primers, hydrolysis probes, and amplicon of the target region are shown in Table 3 below. The initial PCR reaction conditions were 50 cycles consisting of 2 min at 50 캜 and 10 min at 95 캜, followed by 15 sec at 95 캜 and 1 min at 60 캜. A calibration curve was created for each analysis by serial dilution of the ampicillin. All samples were amplified 3 times and the final result reflected the mean. The factor concentration was expressed in copies per mL and a standard factor of 6.6 pg was used to convert the data to the number of copies as described above (Am J Hum Genet 1998; 62: 768 -75).

Marker order X1 SEQ ID NO: 13: Amplicone
5'-AGCTGAAGCTGGGCCGTGGGAAATGTTGAGACGAGTGAGTGCAGAGCCTGAAAGACAATACGGTAGAAAAGGTTACATCCGCG-3 ' SEQ ID NO: 4: forward primer 5'-AGCTGAAGCTGGGCCG-3 '
SEQ ID NO: 5: Reverse primer 5'-CGCGGATGTAACCTTTTCTACCG-3 '
SEQ ID NO: 6: Hydrolysis probe 5 '- (FAM) GAGACGAGTGAGTGCAGAG (MGBNFQ) -3' X2 SEQ ID NO: 14: Amplicone
5'-CCACGTGCACTGAGCGTCTCTTGTTGTTCACCCCAAATAAACTCTGCCGGAACTGGGGCGGGACTCGCAGGGGCGGAGAAGGGGGGAGACGGGCAGAGGGCAGAA-3 ' SEQ ID NO: 7: forward primer 5'-CCACGTGCACTGAGCGTCTC-3 '
SEQ ID NO: 8: Reverse primer 5'-TTCTGCCCTCTGCCCGTCTC-3 '
SEQ ID NO: 9: Hydrolysis probe 5 '- (FAM) CCCAAATAAACTCTGCCGGA (MGBNFQ) -3' X3 SEQ ID NO: 15: Amplicone
5'-CGGCTTTGCCTGAATTAGAGGGCGTGAGAGCCACCTGTGTCTCAGCACTGCAATTAAAGCAGGAAGCCCTTTCGGAAGCAGCCGTGTGCACCA-3 ' SEQ ID NO: 10: forward primer 5'-CGGCTTTGCCTGAATTAGAGG-3 '
SEQ ID NO: 11: Reverse primer 5'-TGGTGCACACGGCTGCT-3 '
SEQ ID NO: 12: Hydrolysis probe 5 '- (FAM) AGGAAGCCCTTTCGG (MGBNFQ) -3'

Figures 4 to 6 show that fetal DNA present in maternal plasma can be effectively detected by qPCR for fetal specific proximal markers X1, X2, and X3.

Example  3. Fetal-Specific Histology Marker  The accuracy of diagnosis of fetal Down syndrome using

The diagnostic accuracy of the Down syndrome fetus was confirmed by using X2 among the fetal specific prognostic markers selected in Example 1 in the samples obtained from the fetal and fetal fetal pregnant blood samples. In order to accurately detect fetal Down syndrome caused by the increase in chromosome number 21 using fetal specific hysteretic markers on chromosome 21 such as X2, the fetal specificity of the reference chromosome with normal chromosome number It is required to confirm the proportionally increased fetal-specific epigenetic markers present on chromosome 21 as compared to methylated and existing epigenetic markers. Therefore, in this embodiment, RASSF1A, a fetal-specific methylation marker corresponding to a region (GeneBank accession No. AP001745; UCSC Genome Browser Assembly: GRCh37 / hg19) on chromosome 3 from 50378107 to 50378202, Respectively. RASSF1A is a fetal-specific methylation marker that is unmethylated in maternal-derived DNA and methylated in placenta-derived DNA (U.S. Patent No. 7754428), UK RAPID for diagnosis of single gene disease using maternal blood- (Reliable Accurate Prenatal Non-Invasive Diagnosis) research project recommends the use of RASSF1A as a hemostatic marker to confirm the presence of fetal DNA in maternal blood.

(1) Isolation of cell-free DNA from maternal plasma

Peripheral blood from pregnant women with Down syndrome (n = 10) and pregnant women with normal fetuses (n = 25) were collected in EDTA tubes and cell- The free DNA was isolated.

(2) Enrichment of methylated DNA

In the same manner as in Example 2, methylated DNA was isolated from circulating DNA extracted from maternal plasma using a MethylMiner ^™ methylated DNA concentration kit (Invitrogen).

(3) multiplex quantitative real-time polymerase chain reaction (multiplex q-RT PCR)

Multiple q-RT PCR was performed to confirm the accuracy of Down syndrome diagnosis using fetal-specific hysteretic markers. 15 [mu] l of 2X Master Mix II (Kogene biotech) and 5 [mu] l of methylated plasma DNA captured by MBD were added to prepare a reaction mixture in a volume of 30 [mu] l. Primers and hydrolysis probes were used at a final concentration of 100 nmol / L. The sequences of the primers, the hydrolysis probe, and the amplicon of the target region are shown in Table 4 below.

The initial PCR reaction conditions were 50 cycles consisting of 2 min at 50 캜 and 10 min at 95 캜, followed by 15 sec at 95 캜 and 1 min at 60 캜. A calibration curve for each assay was generated by serial dilution of the ampicillin. Each sample was amplified three times and the final result reflected the mean. The concentration factor was expressed in copies per mL and a standard factor of 6.6 pg was used to convert the data to the copy number, as described above (Am J Hum Genet 1998; 62: 768-75).

Marker order X2 SEQ ID NO: 14: Amplicone
5'-CCACGTGCACTGAGCGTCTCTTGTTGTTCACCCCAAATAAACTCTGCCGGAACTGGGGCGGGACTCGCAGGGGCGGAGAAGGGGGGAGACGGGCAGAGGGCAGAA-3 ' SEQ ID NO: 7: forward primer 5'-CCACGTGCACTGAGCGTCTC-3 '
SEQ ID NO: 8: Reverse primer 5'-TTCTGCCCTCTGCCCGTCTC-3 '
SEQ ID NO: 9: Hydrolysis probe 5 '- (FAM) CCCAAATAAACTCTGCCGGA (MGBNFQ) -3' RASSF1A SEQ ID NO: 16: Amplicon
5'-AGCTGGCACCCGCTGGGCGCGCTGGGAAGGGCCGCACCCGGCTGGAGCTGCCAACCCGCTGCGCATCGCGCGGGGCACCGCGTGCAACCCCACAC-3 ' SEQ ID NO: 17: forward primer 5'-AGCTGGCACCCGCTGG-3 '
SEQ ID NO: 18: Reverse primer 5'-GTGTGGGGTTGCACGCG-3 '
SEQ ID NO: 19: Hydrolysis probe 5 '- (FAM) ACCCGGCTGGAGCGT (MGBNFQ) -3'

(4) Statistical analysis

The experimental group consisted of pregnant women with Down syndrome pregnancy and the control group consisted of pregnant women with normal pregnancy. X2 and RASSF1A used as a reference marker (the reason for selecting the reference marker ) and the ratio of X2 and RASSF1A were expressed as an average. The t-test was used to compare the difference between the experimental group and the control group Respectively.

The accuracy of detection of fetal Down syndrome was determined based on the concentration of X2 in the blood and the ratio index between X2 and RASSF1A used as a reference marker and referring to the results of fetal karyotype analysis. Where the ratio between X2 and RASSF1A was calculated as (X2 concentration in blood / concentration in RASSF1A blood).

Sensitivity, positive predictive value, negative predictive value, and risk rate were calculated using the EpiMax Table Calculator (http://www.healthstrategy.com/epiperl/epiperl.htm).

The accuracy of the overall diagnostic method was assessed with the area under the ROC curve (AUC). The overall diagnostic accuracy using the area under the ROC curve was calculated for concentrations and ratios in the X2 blood.

Statistical analysis was performed using Statistical Package for Social Sciences 10.0 (SPSS Inc.). Statistical significance was set at P <0.05 for all tests.

As shown in Fig. 7, the amount of RASSF1A used as a reference marker in the maternal blood was not different between the control and experimental groups. However, the amount of X2 in the maternal blood and the ratio of X2 / RASSF1A ratio were significantly increased in the experimental group compared to the control group.

For the diagnostic accuracy of X2 and ratio indices, the cutoff value of each factor was selected at 80% sensitivity by ROC analysis and the specificity, positive predictive value, negative predictive value and AUC were compared. The cutoff value of X2 was 710 copies / mL and showed 52% specificity, 40% positive predictive value, 87% negative predictive value, and 0.716 AUC (95% confidence interval: 0.523-0.909). The cutoff value of ratio index was 3.0, 88% specificity, 73% positive predictive value, 92% negative predictive value, and 0.916 AUC (95% confidence interval: 0.820-1.012). The results of each ROC curve analysis are shown in FIG. In Fig. 8, X2 is indicated as "UMO ", and the rate index is indicated as" RATIO ".

As a result, multiple q-RT PCR results for the fetal specific hysteretic marker X2 and the reference marker RASSF1A indicate that the fetal specific haplotype marker X2 detects fetal DNA present in maternal plasma, To be used for.

&Lt; 110 > CHEIL GENERAL HOSPITAL & WOMEN'S HEALTHCARE CENTER, et al. <120> Fetal Epigenetic Markers and Use thereof <130> PN101107 <160> 19 <170> Kopatentin 2.0 <210> 1 <211> 165 <212> DNA <213> Homo sapiens <400> 1 gccgcacatc agagcagtct atggctgtgg gcttcacagc tgaagctggg ccgtgggaaa 60 tgttgagacg agtgagtgca gagcctgaaa gacaatacgg tagaaaaggt tacatccgcg 120 ggggggcgtc tgatggcatc tgccatttat tgaggatggg tgcga 165 <210> 2 <211> 110 <212> DNA <213> Homo sapiens <400> 2 cacaccccac gtgcactgag cgtctcttgt tgttcacccc aaataaactc tgccggaact 60 ggggcgggac tcgcaggggc ggagaagggg ggagacgggc agagggcaga 110 <210> 3 <211> 167 <212> DNA <213> Homo sapiens <400> 3 ctggtgcacg caaggagcta tcgtgaacat tgctcccaac tggccgcttg cttgtccccc 60 ggctcccctt ggccccagtg gcggctttgc ctgaattaga gggcgtgaga gccacctgtg 120 tctcagcact gcaattaaag caggaagccc tttcggaagc agccgtg 167 <210> 4 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for marker X1 <400> 4 agctgaagct gggccg 16 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for marker X1 <400> 5 cgcggatgta accttttcta ccg 23 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe for marker X1 <400> 6 gagacgagtg agtgcagag 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for marker X2 <400> 7 ccacgtgcac tgagcgtctc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for marker X2 <400> 8 ttctgccctc tgcccgtctc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe for marker X2 <400> 9 cccaaataaa ctctgccgga 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for marker X3 <400> 10 cggctttgcc tgaattagag g 21 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for marker X3 <400> 11 tggtgcacac ggctgct 17 <210> 12 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Probe for marker X3 <400> 12 aggaagccct ttcgg 15 <210> 13 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Amplicon of marker X1 <400> 13 agctgaagct gggccgtggg aaatgttgag acgagtgagt gcagagcctg aaagacaata 60 cggtagaaaa ggttacatcc gcg 83 <210> 14 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Amplicon of marker X2 <400> 14 ccacgtgcac tgagcgtctc ttgttgttca ccccaaataa actctgccgg aactggggcg 60 ggactcgcag gggcggagaa ggggggagac gggcagaggg cagaa 105 <210> 15 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> Amplicon of marker X3 <400> 15 cggctttgcc tgaattagag ggcgtgagag ccacctgtgt ctcagcactg caattaaagc 60 aggaagccct ttcggaagca gccgtgtgca cca 93 <210> 16 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> Amplicon of marker RASSF1A <400> 16 agctggcacc cgctgggcgc gctgggaagg gccgcacccg gctggagcgt gccaacgcgc 60 tgcgcatcgc gcggggcacc gcgtgcaacc ccacac 96 <210> 17 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for RASSF1A <400> 17 agctggcacc cgctgg 16 <210> 18 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for RASSF1A <400> 18 gtgtggggtt gcacgcg 17 <210> 19 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Probe for RASSF1A <400> 19 acccggctgg agcgt 15

Claims (11)

Comprising at least one marker selected from the group consisting of a fetal specific haplotype marker X1 (SEQ ID NO: 1), X2 (SEQ ID NO: 2), and X3 (SEQ ID NO: 3). The composition of claim 1, wherein the composition comprises a primer pair consisting of SEQ ID NOS: 4 and 5 for amplification of marker X1, a probe of SEQ ID NO: 6 for detection of marker X1, and a primer consisting of SEQ ID NOS: 7 and 8 for amplification of marker X2 At least one primer pair selected from the probes of SEQ ID NO: 9 for the detection of the marker X2 and the primer pairs of SEQ ID NOs: 10 and 11 for amplification of the marker X3 and the probe of SEQ ID NO: 12 for the detection of the marker X3, &Lt; / RTI &gt; further comprising a probe. The composition of claim 1, wherein the composition further comprises a buffer, dNTP, and DNA polymerase for real time PCR. A method for detecting fetal DNA in blood of a pregnant woman,
Physically separating the methylated DNA from the maternal blood sample,
Detecting the presence or absence of at least one of fetal-specific haploid markers X1 (SEQ ID NO: 1), X2 (SEQ ID NO: 2), and X3 (SEQ ID NO: 3) for the isolated methylated DNA. 5. The method of claim 4, wherein physically separating the methylated DNA from the pregnant blood is performed by methylated DNA-specific immunoprecipitation or methylated DNA-specific microarrays. 5. The method of claim 4, wherein detecting the presence of one or more of the fetal specific posterior markers X1, X2, and X3 comprises performing real-time PCR. 7. The method of claim 6, wherein the real-time PCR comprises a primer pair consisting of SEQ ID NOS: 4 and 5 for amplification of marker X1, a probe of SEQ ID NO: 6 for detection of marker X1 and SEQ ID NOS: 7 and 8 for amplification of marker X2 At least one primer pair selected from the primer pair, the probe of SEQ ID NO: 9 for detection of marker X2, and the primer pair of SEQ ID NOs: 10 and 11 for amplification of marker X3 and the probe of SEQ ID NO: 12 for detection of marker X3 And a probe. 5. The method of claim 4 wherein detecting the presence of one or more of said fetal specific posterior markers X1, X2, and X3 is performed on a combination of fetal specific posterior markers X1, X2, and X3 . 5. The method of claim 4, wherein said method comprises measuring the level of at least one of fetal-specific prognostic markers X1, X2, and X3 against isolated methylated DNA, and
And comparing the level of the measured marker to a standardized reference for the fetal specific prognostic marker. 10. The method of claim 9, wherein the level of the measured marker relative to a standardized reference to the fetal-specific epigenetic marker provides information necessary for the diagnosis of Down's syndrome or a pregnancy related disorder. The method of claim 9, wherein the standardized criteria for the fetal-specific hysteretic marker include a level of a marker measured from a pregnant woman with a normal fetus, a level of a marker measured from a pregnant woman with a Down syndrome fetus, Wherein the marker is a level of a marker measured from a pregnant woman having a pregnancy related disorder.

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