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WO2025058140A1 - Disease-specific epigenetic biomarker for diagnosing edward syndrome and use thereof - Google Patents

Disease-specific epigenetic biomarker for diagnosing edward syndrome and use thereof Download PDF

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WO2025058140A1
WO2025058140A1 PCT/KR2023/021934 KR2023021934W WO2025058140A1 WO 2025058140 A1 WO2025058140 A1 WO 2025058140A1 KR 2023021934 W KR2023021934 W KR 2023021934W WO 2025058140 A1 WO2025058140 A1 WO 2025058140A1
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gene
cpg
methylation level
methylation
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Korean (ko)
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류현미
임지혜
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Industry Academic Cooperation Foundation of College of Medicine Pochon CHA University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • Edwards syndrome is a genetic disorder caused by a chromosomal abnormality in which there are three copies of chromosome 18. It occurs in 1 in 6,000 to 8,000 newborns, and has many fatal symptoms such as congenital external malformations, developmental delay, mental retardation, heart defects, kidney defects, diaphragmatic hernia, tracheoesophageal fistula, respiratory distress, and muscle weakness. More than 90% of cases die within 6 months of birth, and only about 5% survive to the age of 1.
  • the probability of developing hereditary diseases in the fetus has been rapidly increasing over the past 10 years due to factors such as the aging of the married population and the aging of the childbearing age group.
  • Early diagnosis of genetic abnormalities in the fetus is very important because if diseases containing genetic abnormalities in the fetus are detected in the early stages of pregnancy, it is possible to take prompt medical measures necessary for safety while reducing the suffering of the pregnant woman and the fetus.
  • NGS-based NIPT a method that analyzes quantitative differences in cfDNA, a non-cellular DNA fragment present in maternal plasma, has been developed for prenatal diagnosis and is being rapidly applied in clinical practice.
  • NGS-based NIPT is difficult to apply in general laboratories due to its complex analysis method, and requires expensive analysis equipment and test consumables, resulting in high test costs. This limits its application to all pregnant women.
  • Another prenatal diagnosis method for Edwards syndrome uses SNPs for analysis, so there is no clear basis for test accuracy, and since it requires the use of special algorithms presented by multiple institutions, it has limitations in general application.
  • the inventors of the present invention have discovered the world's first Edwards syndrome-specific biomarker that exhibits significant epigenetic characteristics, and have developed a simple and effective method for diagnosing Edwards syndrome without the need for expensive existing testing devices or special algorithms.
  • One aspect is to provide a composition for diagnosing Edwards syndrome, comprising a preparation for measuring the methylation level of a CpG region of a gene having a CpG region, isolated from a placenta.
  • Another aspect provides the use of a preparation for diagnosing Edwards syndrome, which measures the methylation level of a CpG region of a gene having a CpG region, isolated from the placenta.
  • compositions for diagnosing Edwards syndrome comprising a preparation for measuring the methylation level of a CpG region of a gene having a CpG region, isolated from placenta.
  • Another aspect is to provide a kit for diagnosing Edwards syndrome, comprising the composition.
  • Another aspect provides a method for providing information for diagnosing Edwards syndrome, comprising the steps of: measuring a methylation level of a CpG region of a gene having a CpG region in a placental sample isolated from a subject; and comparing the methylation level measured in the step with the methylation level of the CpG region of the same gene in maternal blood and a normal placenta.
  • Another aspect provides a method for diagnosing Edwards syndrome, comprising the steps of: measuring a methylation level of a CpG region of a gene having a CpG region in a placental sample isolated from a subject; and comparing the methylation level measured in the step with the methylation level of the CpG region of the same gene in maternal blood and a normal placenta.
  • compositions for diagnosing Edwards syndrome comprising a preparation for measuring the methylation level of a CpG region of a gene having a CpG region, isolated from a placenta.
  • Another aspect provides the use of a preparation for diagnosing Edwards syndrome by measuring the methylation level of the CpG region of a gene having a CpG region, isolated from the placenta.
  • compositions for diagnosing Edwards syndrome comprising a preparation for measuring the methylation level of a CpG region of a gene having a CpG region, isolated from placenta.
  • Another aspect provides the utility of a gene with a CpG region isolated from the placenta for the diagnosis of Edwards syndrome.
  • placenta in this specification refers to an organ that connects the fetus and the mother’s endometrium and is responsible for the functions of supplying oxygen and nutrients from the mother, protecting the fetus, and excreting waste from the fetus, and may include the placenta and umbilical cord blood.
  • the placenta may refer to a chorionic villi sample used in CVS (Chorionic Villi Sampling), which is an early prenatal test, rather than the placenta discharged after birth.
  • CpG region in this specification refers to a DNA region of a gene or a part thereof where CpGs are concentrated, called a CpG island.
  • the CpG region may exist in a transcriptional regulatory region such as a promoter region, a protein coding region (open reading frame, ORF), or a terminator region.
  • methylation herein refers to the attachment of a methyl group to a base constituting DNA.
  • the methylation status may refer to the presence or degree of methylation occurring in a cytosine of a specific CpG region of a specific gene. If methylation occurs, the binding of a transcription factor may be interfered with, thereby inhibiting the expression of the corresponding gene. The expression inhibition may occur relatively depending on the degree of methylation. Conversely, if unmethylation or hypomethylation occurs, the expression of a specific gene may increase.
  • DNA methylation is an epigenetic modification that plays a pivotal role in the regulation of gene expression, in which a methyl group (-CH3) is attached to the 5th carbon of cytosine, thereby transforming it into the form of 5-methylcytosine.
  • DNA methylation mainly occurs in the cytosine group of a CpG dinucleotide, and is frequently found in DNA regions where CpG is concentrated, called CpG islands or CpG regions.
  • the gene may comprise one or more genes selected from the group consisting of (a) and (b):
  • the gene of (a) can be selected from Table 1 below.
  • the gene of (b) can be selected from Table 2 below.
  • gene A/gene B means a region existing between two genes, and specifically means a region between two genes excluding the regions of the two genes. That is, “gene A/gene B” does not include the region of gene A and the region of gene B, but includes the region between gene A and gene B.
  • a fragment having a CpG region of the gene may mean a fragment having selectivity and specificity for the gene so as to represent the gene among fragments having the CpG region of the gene. Accordingly, by measuring the methylation level of a fragment having a CpG region of any gene, the methylation level of the gene can be compared, and Edwards syndrome can be diagnosed accordingly.
  • any gene since the location of the CpG region of any gene is known in the art, those skilled in the art can appropriately select a fragment having a CpG region in the gene to realize the desired effect of the present invention, or can utilize a fragment having a CpG region known in the art to represent any gene.
  • a preparation for measuring the methylation level of such a fragment can be purchased to compare the methylation level of the desired gene.
  • the gene or a fragment having a CpG region of the gene may be a gene or a fragment thereof having a region where two or more epigenetic characteristics in which the methylation level of the CpG region increases or decreases sequentially in the order of the mother, a normal fetus, and an Edwards syndrome fetus exist, and where the epigenetic characteristics are maintained.
  • level measurement herein may include measuring the degree of methylation of a marker for Edwards syndrome in a biological sample to diagnose Edwards syndrome. In addition, it may include measuring the degree of expression or activity of said marker.
  • the term "measuring the methylation level of a CpG region” in the present specification may mean measuring the methylation level of a CpG region of the gene, and may be performed by at least one selected from the group consisting of, but not limited to, Methyl capture sequencing (MC-seq), Polymerase Chain Reaction (PCR), methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA-specific binding protein, methyl PNA-PCR, quantitative PCR, DNA chip, pyrosequencing, and bisulfite sequencing.
  • MC-seq Methyl capture sequencing
  • PCR Polymerase Chain Reaction
  • methylation specific PCR methylation specific PCR
  • real time methylation specific PCR PCR using methylated DNA-specific binding protein
  • methyl PNA-PCR quantitative PCR
  • DNA chip DNA chip
  • pyrosequencing pyrosequencing
  • the measurement of the methylation level of the above CpG region may be measured at the genome level.
  • marker refers to a substance that can be evaluated to distinguish between individuals having an immune-related disease and individuals not having an immune-related disease, and may include organic biomolecules such as polypeptides, nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), metabolites, etc., which show an increase or decrease in individuals having an immune-related disease compared to individuals not having the immune-related disease.
  • nucleic acids e.g., mRNA, etc.
  • lipids e.g., glycolipids, glycoproteins
  • sugars monosaccharides, disaccharides, oligosaccharides, etc.
  • metabolites etc.
  • diagnosis herein may refer to determining a subject's susceptibility to a particular disease or condition, determining whether a subject has a particular disease or condition, determining a prognosis for a subject having a particular disease or condition, or monitoring the same.
  • Edwards syndrome in this specification refers to a genetic disorder (trisomy genetic disorder) caused by a chromosomal abnormality in which there are three copies of chromosome 18.
  • the above Edwards syndrome pathologies may include congenital external malformations, externally small and narrow head, occipital prominence, microstomia, cleft lip and palate, micrognathia, short sternum, small pelvis, characteristic hand and foot shapes (index finger over middle finger and little finger over fourth finger), rocker-bottom feet, short and backward-bent first toes, developmental delay, mental retardation, severe mental retardation, heart malformations (ventricular septal defect, patent ductus arteriosus), renal malformations (horsehair kidney), diaphragmatic hernia, tracheoesophageal fistula, respiratory distress, and muscle weakness.
  • the agent for measuring the methylation level of the CpG region of the gene may include bisulfite or a salt thereof, a methylation-sensitive restriction enzyme, a primer that specifically binds to a sequence including a CpG site of the gene, a probe that can hybridize to a sequence including a methylated CpG site of the gene, a methylated CpG binding domain, or an antibody or aptamer that specifically binds to methylcytosine.
  • Another aspect provides a kit for diagnosing Edwards syndrome, comprising the composition.
  • the above Edwards syndrome is as described above.
  • the kit may comprise (i) a plurality of primers, probes or antisense nucleotides capable of hybridizing to a gene specific for diagnosing Edwards syndrome, or a fragment thereof, as described above; (ii) a container suitable for containing a plurality of primers, probes or antisense nucleotides capable of hybridizing to the gene or a fragment thereof and a biological sample of the subject (e.g., a nucleic acid isolated from the biological sample); (iii) means for detecting the hybridization of (ii); and/or optionally (iv) instructions for use and interpretation of the kit results.
  • the above kit may also contain other components, including a hybridization solution packaged in a separate container, in which the nucleic acid of the gene can be hybridized with a plurality of primers, probes or antisense nucleotides.
  • the kit may be composed of one or more types of other component compositions, solutions or devices suitable for the analysis method.
  • the kit may be an RT-PCR (Reverse transcription polymerase chain reaction) kit, a DNA chip kit.
  • the kit may additionally include a polymerase agarose, a buffer solution required for electrophoresis, etc.
  • the kit may include a diagnostic nucleic acid chip (CHIP) or gene panel having a probe immobilized thereon, which is capable of hybridizing with a gene having the CpG region or a fragment thereof having the CpG region.
  • a diagnostic nucleic acid chip (CHIP) or gene panel having a probe immobilized thereon, which is capable of hybridizing with a gene having the CpG region or a fragment thereof having the CpG region.
  • the kit may include a computer having a built-in agent, device, and algorithm for measuring the methylation level of a CpG region of the gene or a fragment thereof, and may relate to a kit that associates the result of measuring the level of the marker with a diagnosis of Edwards syndrome through the algorithm.
  • Another aspect provides a method for providing information for diagnosing Edwards syndrome, comprising the steps of: measuring a methylation level of a CpG region of a gene having a CpG region in a placental sample isolated from a subject; and comparing the methylation level measured in the step with the methylation level of the CpG region of the same gene in maternal blood and a normal placenta.
  • Another aspect provides a method for diagnosing Edwards syndrome, comprising the steps of: measuring a methylation level of a CpG region of a gene having a CpG region in a placental sample isolated from a subject; and comparing the methylation level measured in the step with the methylation level of the CpG region of the same gene in maternal blood and a normal placenta.
  • the placenta may refer to a chorionic villi sample used in CVS (Chorionic Villi Sampling), which is an early prenatal test, rather than a placenta discharged after birth.
  • CVS Chorionic Villi Sampling
  • the step of measuring the methylation level may be performed by at least one selected from the group consisting of PCR, methylation-specific PCR, real time methylation-specific PCR, MethyLight PCR, MehtyLight digital PCR, EpiTYPER, PCR using methylated DNA-specific binding protein, methyl PNA-PCR, quantitative PCR, DNA chip, pyrosequencing, bisulfite sequencing, Southern blot, RLGS (Restriction landmark genomic scanning), MS-SnuPE (Methylation-sensitive single-nucleotide primer extension), CpG microarray, COBRA (Combined bisulfite-restriction analysis), MIRA (methylated-CpG island recovery assay), mass spectrometry, and immunoprecipitation using a methylated CpG binding domain or an anti-methylcytosine antibody.
  • PCR methylation-specific PCR
  • real time methylation-specific PCR MethyLight PCR
  • the gene may comprise one or more genes selected from the group consisting of (a) and (b):
  • the gene of (a) can be selected from Table 1.
  • the gene of (b) can be selected from Table 2.
  • the method for providing information for diagnosing Edwards syndrome may further include a step of determining Edwards syndrome when the methylation level of the gene of (a) is increased compared to the methylation level of the CpG region of the same gene in maternal blood and normal placenta, or determining Edwards syndrome when the methylation level of the gene of (b) is decreased compared to the methylation level of the CpG region of the same gene in maternal blood and normal placenta.
  • methylation level of a gene or “increased methylation level of a CpG region of a gene” as used herein means that the methylation level in a sample of a subject is measurably and significantly increased compared to a control, and may mean, for example, an increase of about 1.1-fold or more, for example, 1.1-fold to 2.5-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold or more.
  • decreased methylation level of a gene or “decreased methylation level of a CpG region of a gene” as used herein means a measurable and significant decrease in the methylation level in a sample of a subject compared to a control, for example, about 0.9-fold or less, for example, 0.1-fold to 0.9-fold, 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, or 0.1-fold or less.
  • the method for diagnosing Edwards syndrome can be performed using a plurality of panels.
  • a composition for diagnosing Edwards syndrome according to one aspect or a method for providing information for diagnosing the same can simply and effectively diagnose Edwards syndrome without the need for expensive existing testing devices or special algorithms by measuring and comparing the methylation level of disease-specific epigenetic biomarkers that change in a patient.
  • Figures 1 and 2 show the results of a heat map analysis of the levels of Edwards syndrome-specific hypermethylation biomarkers selected for the diagnosis of Edwards syndrome.
  • Figures 3 and 4 show the results of a heat map analysis of the levels of Edwards syndrome-specific hypomethylated biomarkers selected for the diagnosis of Edwards syndrome.
  • a and/or B means A or B, or A and B.
  • Genomes were extracted from blood samples from five maternal cases.
  • DNeasy Blood & Tissue mini kits from Qiagen were used according to the manufacturer's instructions, and the detailed methods are as follows. 20 ⁇ l of proteinase K, 20 ⁇ l of RNase, 200 ⁇ l of sample, and 200 ⁇ l of AL buffer were added to a 1.5 ml tube from Axygen, mixed, and incubated at 56 °C for 20 minutes. Then, 100% ethanol was added to the sample, mixed, and the mixture was transferred to a DNeasy Mini spin column and centrifuged at 8,000 rpm for 1 minute. For purification, 500 ml of Buffer AW1 was added, centrifuged, and then 500 ⁇ l of Buffer AW2 was added, centrifuged again. The purified sample was recovered using the DNeasy Mini spin column, and 50 ⁇ l of buffer AE was added to base-convert the DNA, and then recovered. Finally, the purification steps were repeated to complete the work until the total volume became 100 ⁇ l.
  • Genomes were extracted from placental samples from five normal fetuses and five Edwards syndrome fetuses.
  • the tissues were placed in 1.5 ml microtubes (Axygen), 3 mm tungsten carbide beads, 20 ⁇ l of proteinase K, and 200 ⁇ l of ATL buffer were added, and the Tissuelyzer was operated at 25 Hz for 3 minutes. Incubation was then performed at 56°C for 20 minutes. Subsequent processes were performed using Qiagen's DNeasy Blood & Tissue mini kits according to the manufacturer's instructions, and the detailed methods are as follows. After adding 20 ⁇ l of RNase and 200 ⁇ l of AL buffer, the tube was mixed thoroughly. 100% ethanol was added to the sample and mixed thoroughly.
  • the mixture was transferred to a DNeasy Mini spin column, centrifuged at 8,000 rpm for 1 minute, and the collection tube was replaced with a new one. After adding 500 ⁇ l of Buffer AW1, centrifugation was performed at 8000 rpm for 1 minute, and the collection tube was replaced with a new one. Similarly, after adding 500 ⁇ l of Buffer AW2, centrifugation was performed at 8000 rpm for 1 minute, the flow-through was discarded, and the DNeasy Mini spin column was reinserted into the collection tube. After centrifugation at maximum speed for 1 minute, the DNeasy Mini spin column was placed in a new 1.5 ml tube. After adding 50 ⁇ l of Buffer AE, incubation was performed at room temperature for 1 minute, and centrifugation was performed at maximum speed for 1 minute.
  • MC-seq methyl capture sequencing
  • the methylation level of the CpG region of a gene or fragment thereof having a CpG region was analyzed using MC-seq.
  • MC-seq libraries were prepared using the SureSelectXT Methyl-Seq Library Preparation kit (Agilent Technologies, Germany).
  • 3 mg of genomic DNA is fragmented to 150–200 bp using a Covaris LE220 focused-ultrasonicator (Covaris, Woburn, MA), and SureSelect Methyl-Seq Methylated Adapters are ligated to the sheared DNA fragments.
  • the methylated adapter-ligated DNA is quantified using TapeStation DNA screentape D1000 (Agilent).
  • 350 ng of DNA library is mixed with mixing buffer, blocking mixture, RNase block, and 5 ⁇ L of SureSelect Methyl-Seq Capture Library according to the Agilent SureSelectXT Methyl-Seq Target Enrichment protocol.
  • Hybridization to the Capture baits is performed at 65°C for 24 h using a thermocycler lid option heated at 105°C in a PCR machine.
  • the captured methyl DNA is then washed and amplified, and the final purified DNAs are quantified using qPCR according to the qPCR quantification protocol guide (KAPA Library Quantification Kit for Illumina Sequencing Platforms) and qualitatively verified using TapeStation DNA Screen Tape HSD1000 (Agilent). They are then sequenced using the NovaSeq platform (Illumina, San Diego, USA).
  • the refined reads are aligned to the human genome (UCSC hg19) using BSMAP, and BSMAP (version 2.90 parameter setting -n 1 -r 0) allows only uniquely mapped reads, and the mapped data (SAM file format) is aligned and indexed via sambamba (version 0.6.5). After that, PCR duplicates are removed using sambamba (version 0.6.5). Methylation levels are called using methratio.py from the BSMAP program, which performs methylation calling of all single cytosines located in the Agilent SureSelect target region, and the resulting coverage profiles are interpreted as # of C/valid CT counts for each of the three sequence contexts (CG, CHG, and CHH).
  • the methylation level of a CpG site is expressed as 0 to 100, with 0 indicating unmethylation and 100 indicating complete methylation.
  • biomarkers were discovered by dividing them into hypermethylated markers and hypomethylated markers according to the epigenetic characteristics shown in the placenta of Edwards syndrome fetuses. Regions where two or more epigenetic characteristics of the same type exist consecutively, including the marker region, were selected. Specifically, a gene or a fragment thereof having a region where the methylation level of the CpG region increases or decreases in the order of the mother, normal fetus, and Edwards syndrome fetus, and where the epigenetic characteristics are maintained, were selected as biomarkers.
  • the selected hypermethylation biomarker shows a low methylation level of 0.2 or less in the maternal blood, a high hypermethylation of ⁇ + 0.2 to + 0.3 in the normal fetus compared to the maternal blood, and a high hypermethylation of ⁇ + 0.2 to + 0.3 in the Edwards syndrome fetus compared to the normal fetus.
  • the 67 selected hypermethylation biomarkers are shown in Table 3 below.
  • methylation levels (methylation level (%) divided by 100) of the selected gene regions in normal maternal blood, normal fetal placenta, and Edwards syndrome fetal placenta are shown in Figures 1, 2, and Table 4 below.
  • Figures 1 and 2 show the results of a heat map analysis of the levels of Edwards syndrome-specific hypermethylation biomarkers selected for the diagnosis of Edwards syndrome.
  • NB represents maternal blood
  • NT represents the placenta of a normal fetus
  • T18T represents the placenta of an Edwards syndrome fetus.
  • the above 67 genes were selected as hypermethylation biomarkers, and the methylation level of the CpG site in the above biomarkers was about 10 to 50 higher in the normal fetal placenta than in the maternal blood, and about 10 to 50 higher in the Edwards syndrome fetal placenta than in the normal fetal placenta.
  • a gene or a fragment thereof that has a lower methylation level in the normal fetal placenta than in the maternal blood and a lower methylation level in the Edwards syndrome fetal placenta than in the normal fetal placenta was selected as a hypomethylation biomarker.
  • the selected hypomethylation biomarker shows a high methylation level of 0.8 or higher in the maternal blood, a low hypomethylation of ⁇ - 0.4 to - 0.5 in the normal fetus compared to the maternal blood, and a low hypomethylation of ⁇ - 0.2 to - 0.4 in the Edwards syndrome fetus compared to the normal fetus.
  • the 91 selected hypomethylated biomarkers are shown in Table 5 below.
  • methylation levels (methylation level (%) divided by 100) of the selected gene regions in normal maternal blood, normal fetal placenta, and Edwards syndrome fetal placenta are shown in Figures 3 and 4 and Table 6 below.
  • Figures 3 and 4 show the results of a heat map analysis of the levels of Edwards syndrome-specific hypomethylated biomarkers selected for the diagnosis of Edwards syndrome.
  • NB represents maternal blood
  • NT represents the placenta of a normal fetus
  • T18T represents the placenta of an Edwards syndrome fetus.
  • the above 91 genes were selected as hypomethylated biomarkers, and the methylation level of the CpG site in the above biomarkers was about 50 to 75 lower in the normal fetal placenta than in the maternal blood, and about 10 to 50 lower in the Edwards syndrome fetal placenta than in the normal fetal placenta.

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Abstract

The present invention relates to a disease-specific epigenetic biomarker for diagnosing Edward syndrome and a use thereof. A composition for diagnosing Edward syndrome or a method for providing information for diagnosing same according to an aspect of the present invention involve measuring and comparing a methylation level of a disease-specific epigenetic biomarker changing in a patient, thereby simply and effectively diagnosing Edward syndrome without the need for existing expensive diagnostic devices or a special algorithm.

Description

에드워드 증후군 진단을 위한 질환 특이적인 후성학적 바이오마커 및 그의 용도Disease-specific epigenetic biomarkers for diagnosing Edwards syndrome and their uses

에드워드 증후군 진단을 위한 질환 특이적인 후성학적 바이오마커 및 그의 용도에 관한 것이다.Disease-specific epigenetic biomarkers and their uses for diagnosing Edwards syndrome.

에드워드 증후군은 18번 염색체가 3개 존재하는 염색체 이상으로 유발되는 유전 질환으로, 신생아 6,000 내지 8,000명 중 1명의 빈도로 나타나며, 선천적 외형 기형, 발달 지연, 정신 지체, 심장 기형, 신장 기형, 횡경막 탈장, 기관 식도루, 호흡 장애, 근육 약화 등의 치명적인 증상이 많아 90% 이상이 생후 6개월 이내 사망하고 5% 정도만 1세까지 생존한다.Edwards syndrome is a genetic disorder caused by a chromosomal abnormality in which there are three copies of chromosome 18. It occurs in 1 in 6,000 to 8,000 newborns, and has many fatal symptoms such as congenital external malformations, developmental delay, mental retardation, heart defects, kidney defects, diaphragmatic hernia, tracheoesophageal fistula, respiratory distress, and muscle weakness. More than 90% of cases die within 6 months of birth, and only about 5% survive to the age of 1.

결혼 인구의 고령화 및 출산 연령대의 고령화 등의 원인으로 태아의 유전성 질병 발생 확률이 최근 10년간 빠르게 증가하고 있다. 태아의 유전적 이상을 포함하는 질환을 임신 초기에 검출할 경우, 임부와 태아에게 고통을 줄이면서도 안전에 필요한 조속한 의학적 조치가 가능하기 때문에, 태아의 유전적 이상을 조기에 진단하는 것은 매우 중요하다.The probability of developing hereditary diseases in the fetus has been rapidly increasing over the past 10 years due to factors such as the aging of the married population and the aging of the childbearing age group. Early diagnosis of genetic abnormalities in the fetus is very important because if diseases containing genetic abnormalities in the fetus are detected in the early stages of pregnancy, it is possible to take prompt medical measures necessary for safety while reducing the suffering of the pregnant woman and the fetus.

현재 에드워드 증후군의 경우, 산전 진단으로 임산부 혈장에 존재하는 비세포성 DNA 조각인 cfDNA의 양적 차이를 분석하는 방법(NGS based NIPT)이 개발되었으며 임상에 빠르게 적용되고 있다. 하지만, NGS based NIPT은 분석 방법이 복잡하여 일반 검사실에서 적용하기 어렵고, 고가의 분석 기기와 검사 소모품이 필요하여 고가의 검사 비용이 요구된다. 이로 인해, 모든 임산부에 적용하는 것에 한계가 있다.Currently, in the case of Edwards syndrome, a method (NGS-based NIPT) that analyzes quantitative differences in cfDNA, a non-cellular DNA fragment present in maternal plasma, has been developed for prenatal diagnosis and is being rapidly applied in clinical practice. However, NGS-based NIPT is difficult to apply in general laboratories due to its complex analysis method, and requires expensive analysis equipment and test consumables, resulting in high test costs. This limits its application to all pregnant women.

다른 에드워드 증후군 산전 진단 방법으로 PCR based NIPT는 분석방법이 SNP를 이용하므로 검사 정확성에 대한 명확한 근거가 없으며, 여러 기관에서 제시하는 특수 알고리즘을 사용해야 하므로 범용적으로 적용하는 데에 한계가 있다.Another prenatal diagnosis method for Edwards syndrome, PCR-based NIPT, uses SNPs for analysis, so there is no clear basis for test accuracy, and since it requires the use of special algorithms presented by multiple institutions, it has limitations in general application.

이에 따라 종래의 기술상 문제를 해결하며 에드워드 증후군을 효과적으로 진단할 수 있는 방법이 필요한 실정이다.Accordingly, there is a need for a method that can effectively diagnose Edwards syndrome while solving the problems of conventional technology.

이에 본 발명자들은 유의한 후성학적 특징을 나타내는 세계 최초의 에드워드 증후군 특이적인 바이오마커를 발굴하였으며, 기존 고가의 검사 기기 혹은 특수 알고리즘의 필요없이 간단하고 효과적으로 에드워드 증후군을 진단할 수 있는 방법을 도출하였다. Accordingly, the inventors of the present invention have discovered the world's first Edwards syndrome-specific biomarker that exhibits significant epigenetic characteristics, and have developed a simple and effective method for diagnosing Edwards syndrome without the need for expensive existing testing devices or special algorithms.

일 양상은 태반으로부터 분리된, CpG 영역을 갖는 유전자의 CpG 영역의 메틸화 수준을 측정하는 제제를 포함하는, 에드워드증후군 진단용 조성물을 제공하는 것이다.One aspect is to provide a composition for diagnosing Edwards syndrome, comprising a preparation for measuring the methylation level of a CpG region of a gene having a CpG region, isolated from a placenta.

다른 양상은 태반으로부터 분리된, CpG 영역을 갖는 유전자의 CpG 영역의 메틸화 수준을 측정하는 제제의 에드워드증후군 진단을 위한 용도를 제공하는 것이다.Another aspect provides the use of a preparation for diagnosing Edwards syndrome, which measures the methylation level of a CpG region of a gene having a CpG region, isolated from the placenta.

또 다른 양상은 태반으로부터 분리된, CpG 영역을 갖는 유전자의 CpG 영역의 메틸화 수준을 측정하는 제제의 에드워드증후군 진단용 조성물의 제조를 위한 용도를 제공하는 것이다.Another aspect provides a use for the preparation of a composition for diagnosing Edwards syndrome, the composition comprising a preparation for measuring the methylation level of a CpG region of a gene having a CpG region, isolated from placenta.

또 다른 양상은 상기 조성물을 포함하는, 에드워드증후군 진단용 키트를 제공하는 것이다.Another aspect is to provide a kit for diagnosing Edwards syndrome, comprising the composition.

또 다른 양상은 대상체로부터 분리된 태반시료에서 CpG 영역을 갖는 유전자의 CpG 영역의 메틸화 수준을 측정하는 단계; 및 상기 단계에서 측정된 메틸화 수준을 모체 혈액 및 정상 태반 내 동일 유전자의 CpG 영역의 메틸화 수준과 비교하는 단계를 포함하는, 에드워드증후군의 진단을 위한 정보를 제공하는 방법을 제공하는 것이다.Another aspect provides a method for providing information for diagnosing Edwards syndrome, comprising the steps of: measuring a methylation level of a CpG region of a gene having a CpG region in a placental sample isolated from a subject; and comparing the methylation level measured in the step with the methylation level of the CpG region of the same gene in maternal blood and a normal placenta.

또 다른 양상은 대상체로부터 분리된 태반시료에서 CpG 영역을 갖는 유전자의 CpG 영역의 메틸화 수준을 측정하는 단계; 및 상기 단계에서 측정된 메틸화 수준을 모체 혈액 및 정상 태반 내 동일 유전자의 CpG 영역의 메틸화 수준과 비교하는 단계를 포함하는, 에드워드증후군의 진단 방법을 제공하는 것이다.Another aspect provides a method for diagnosing Edwards syndrome, comprising the steps of: measuring a methylation level of a CpG region of a gene having a CpG region in a placental sample isolated from a subject; and comparing the methylation level measured in the step with the methylation level of the CpG region of the same gene in maternal blood and a normal placenta.

일 양상은 태반으로부터 분리된, CpG 영역을 갖는 유전자의 CpG 영역의 메틸화 수준을 측정하는 제제를 포함하는, 에드워드증후군 진단용 조성물을 제공한다.One aspect provides a composition for diagnosing Edwards syndrome, comprising a preparation for measuring the methylation level of a CpG region of a gene having a CpG region, isolated from a placenta.

다른 양상은 태반으로부터 분리된, CpG 영역을 갖는 유전자의 CpG 영역의 메틸화 수준을 측정하는 제제의 에드워드증후군 진단을 위한 용도를 제공한다.Another aspect provides the use of a preparation for diagnosing Edwards syndrome by measuring the methylation level of the CpG region of a gene having a CpG region, isolated from the placenta.

또 다른 양상은 태반으로부터 분리된, CpG 영역을 갖는 유전자의 CpG 영역의 메틸화 수준을 측정하는 제제의 에드워드증후군 진단용 조성물의 제조를 위한 용도를 제공한다.Another aspect provides a use for the preparation of a composition for diagnosing Edwards syndrome, comprising a preparation for measuring the methylation level of a CpG region of a gene having a CpG region, isolated from placenta.

또 다른 양상은 태반으로부터 분리된, CpG 영역을 갖는 유전자의 에드워드증후군 진단을 위한 용도를 제공한다.Another aspect provides the utility of a gene with a CpG region isolated from the placenta for the diagnosis of Edwards syndrome.

본 명세서에서 용어 “태반”은 태아와 모체의 자궁내막을 연결하여 모체로부터 산소와 영양분 공급, 태아 보호, 태아의 노폐물 배출 등의 기능을 담당하는 기관으로, 태반 및 제대혈(cord blood)을 포함할 수 있다. 또한, 상기 태반은 출산 후 배출되는 태반이 아닌, 초기산전검사인 CVS(Chorionic Villi Sampling)에 사용되는 융모막 검체를 의미할 수 있다.The term “placenta” in this specification refers to an organ that connects the fetus and the mother’s endometrium and is responsible for the functions of supplying oxygen and nutrients from the mother, protecting the fetus, and excreting waste from the fetus, and may include the placenta and umbilical cord blood. In addition, the placenta may refer to a chorionic villi sample used in CVS (Chorionic Villi Sampling), which is an early prenatal test, rather than the placenta discharged after birth.

본 명세서에서 용어 "CpG 영역"은 CpG 섬이라고 불리는 CpG가 집중적으로 몰려있는 유전자의 DNA 영역 또는 그의 일부를 지칭한다. 예컨대, 상기 CpG 영역은 프로모터 영역 등 전사 조절 영역, 단백질 코딩 영역(open reading frame, ORF) 또는 터미네이터 영역 등에 존재할 수 있다. The term "CpG region" in this specification refers to a DNA region of a gene or a part thereof where CpGs are concentrated, called a CpG island. For example, the CpG region may exist in a transcriptional regulatory region such as a promoter region, a protein coding region (open reading frame, ORF), or a terminator region.

본 명세서에서 용어 "메틸화"는 DNA를 구성하는 염기에 메틸기가 부착되는 것을 의미한다. 일 구체예에서, 메틸화 상태는 특정 유전자의 특정 CpG 영역의 사이토신에서 일어나는 메틸화 여부 또는 메틸화 정도를 의미할 수 있다. 메틸화가 일어난 경우 그로 인하여 전사인자의 결합이 방해를 받아 해당 유전자의 발현이 억제될 수 있다. 발현 억제는 메틸화 정도에 따라 상대적으로 일어날 수 있다. 반대로, 비메틸화 또는 저메틸화가 일어나는 경우 특정 유전자의 발현이 증가할 수 있다. 이러한 이유로 DNA 메틸화는 유전자 발현 조절에 중추적인 역할을 하는 후성유전적 변형으로서, 사이토신의 5번 탄소에 메틸기(-CH3)가 붙어 5-메틸사이토신 형태로 변형된다. DNA 메틸화는 주로 CpG 디뉴클레오티드의 사이토신 기에서 발생하며, CpG 섬 또는 CpG 영역이라고 불리는 CpG가 집중적으로 몰려 있는 DNA 영역에서 많이 나타난다.The term "methylation" herein refers to the attachment of a methyl group to a base constituting DNA. In one specific example, the methylation status may refer to the presence or degree of methylation occurring in a cytosine of a specific CpG region of a specific gene. If methylation occurs, the binding of a transcription factor may be interfered with, thereby inhibiting the expression of the corresponding gene. The expression inhibition may occur relatively depending on the degree of methylation. Conversely, if unmethylation or hypomethylation occurs, the expression of a specific gene may increase. For this reason, DNA methylation is an epigenetic modification that plays a pivotal role in the regulation of gene expression, in which a methyl group (-CH3) is attached to the 5th carbon of cytosine, thereby transforming it into the form of 5-methylcytosine. DNA methylation mainly occurs in the cytosine group of a CpG dinucleotide, and is frequently found in DNA regions where CpG is concentrated, called CpG islands or CpG regions.

일 구체예에 있어서, 상기 유전자는 하기 (a) 및 (b)로 이루어진 군에서 선택되는 하나 이상의 유전자를 포함하는 것일 수 있다:In one specific example, the gene may comprise one or more genes selected from the group consisting of (a) and (b):

(a) MXRA8, PTPRU, IL12RB2, WNT3A, KIF26B, SLC30A3/DNAJC5G, ZFP36L2, ZIC4, CPLX1, ZFYVE28, CCNG2/CXCL13, PALLD, IRX4, C5orf38, ISL1, LHFPL2, GPRIN1, LOC100506207/TFAP2A, GFOD1/SIRT5, ETV7, FOXP4, SMOC2, GNA12/CARD11, AP5Z1, ZNF273/ZNF117, CALN1, SND1, HTR5A/INSIG1, MNX1, EBF2/PPP2R2A, FAM110B, OSR2/VPS13B, NAPRT, GLIS3, SLC24A2/MLLT3, CRB2, RABL6, TACC2, CPXM2/CHST15, CLMP, KRT7/KRT81, HOXC4, LOC100287944, LINC00403, LINC00925, NR2F2-AS1, MGRN1, RBFOX1, PLEKHH3, EMILIN2, ST8SIA3, ST8SIA3/ONECUT2, ONECUT2, NFATC1/CTDP1, PARD6G, ZNF563, MED29/ZFP36, DYRK1B, PPP1R13L, CYP24A1, C21orf2/TRPM2, YBEY,및 MAPK8IP2 및 이들의 CpG 영역을 갖는 단편; 및 (a) MXRA8, PTPRU, IL12RB2, WNT3A, KIF26B, SLC30A3/DNAJC5G, ZFP36L2, ZIC4, CPLX1, ZFYVE28, CCNG2/CXCL13, PALLD, IRX4, C5orf38, ISL1, LHFPL2, GPRIN1, LOC100506207/TFAP2A, GFOD1/SIRT5, ETV7, FOXP4, SMOC2, GNA12/CARD11, AP5Z1, ZNF273/ZNF117, CALN1, SND1, HTR5A/INSIG1, MNX1, EBF2/PPP2R2A, FAM110B, OSR2/VPS13B, NAPRT, GLIS3, SLC24A2/MLLT3, CRB2, RABL6, TACC2, CPXM2/CHST15, CLMP, KRT7/KRT81, HOXC4, LOC100287944, LINC00403, LINC00925, NR2F2-AS1, MGRN1, RBFOX1, PLEKHH3, EMILIN2, ST8SIA3, ST8SIA3/ONECUT2, ONECUT2, NFATC1/CTDP1, PARD6G, ZNF563, MED29/ZFP36, DYRK1B, PPP1R13L, CYP24A1, C21orf2/TRPM2, YBEY, and MAPK8IP2 and fragments having their CpG regions; and

(b) LCK, FCRL5, RHOU/RAB4A, GREM2/RGS7, DCDC2C/LINC01304, RNU6-81P, CCDC108, LOC150935/MIR4786, AQP12B, AQP12A, LINC01237, XCR1, FGFR3, NDST4/MIR1973, TPPP, HNRNPH1/C5orf60, RASGEF1C, LINC01012/LOC100131289, HLA-DPA1, SMOC2/THBS2, ADAP1, ELFN1, MAD1L1, LYPD2, MAPK15, PUF60, LINGO2/LINC01242, APBA1, NCS1, FIBCD1, RXRA/COL5A1, KCNT1, GPSM1, PFKFB3, ATRNL1, FRG2B/DUX4L3, PKP3, MUC5B, LMO1, LOC441601, APOA5, TRIM29, B4GALNT3, CACNA2D4/LRTM2, TSPAN9/PRMT8, VAMP1/MRPL51, GNB3, LRIG3/SLC16A7, CABP1, TMEM132D, LINC01257, LOC101928416/FBRSL1, LINC00572, MCF2L, GRTP1/ADPRHL1, EAPP/SNX6, ELK2AP/KIAA0125, ADAM6/LINC00226, LINC00226/LINC00221, C15orf48, SV2B, LOC101927332/FAM169B, SBK1, RRN3P2/SNX29P2, ARHGEF15, NFE2L1, SEC14L1/SEPT9, B3GNTL1/METRNL, AQP4-AS1, TSHZ1, GALR1/LINC01029, LINC01029/SALL3, GRIN3B, SMIM24/DOHH, C19orf67, TMC4, LILRB3, SIRPG/LOC100289473, FOXS1, HELZ2, LOC101927797/LINC00320, LINC00111, TFF2, ZDHHC8P1, LOC284930 및 이들의 CpG 영역을 갖는 단편.(b) LCK, FCRL5, RHOU/RAB4A, GREM2/RGS7, DCDC2C/LINC01304, RNU6-81P, CCDC108, LOC150935/MIR4786, AQP12B, AQP12A, LINC01237, XCR1, FGFR3, NDST4/MIR1973, TPPP, HNRNPH1/C5orf60, RASGEF1C, LINC01012/LOC100131289, HLA-DPA1, SMOC2/THBS2, ADAP1, ELFN1, MAD1L1, LYPD2, MAPK15, PUF60, LINGO2/LINC01242, APBA1, NCS1, FIBCD1, RXRA/COL5A1, KCNT1, GPSM1, PFKFB3, ATRNL1, FRG2B/DUX4L3, PKP3, MUC5B, LMO1, LOC441601, APOA5, TRIM29, B4GALNT3, CACNA2D4/LRTM2, TSPAN9/PRMT8, VAMP1/MRPL51, GNB3, LRIG3/SLC16A7, CABP1, TMEM132D, LINC01257, LOC101928416/FBRSL1, LINC00572, MCF2L, GRTP1/ADPRHL1, EAPP/SNX6, ELK2AP/KIAA0125, ADAM6/LINC00226, LINC00226/LINC00221, C15orf48, SV2B, LOC101927332/FAM169B, SBK1, RRN3P2/SNX29P2, ARHGEF15, NFE2L1, SEC14L1/SEPT9, B3GNTL1/METRNL, AQP4-AS1, TSHZ1, GALR1/LINC01029, LINC01029/SALL3, GRIN3B, SMIM24/DOHH, C19orf67, TMC4, LILRB3, SIRPG/LOC100289473, FOXS1, HELZ2, LOC101927797/LINC00320, LINC00111, TFF2, ZDHHC8P1, LOC284930 and fragments having their CpG regions.

일 구체예에 있어서, 상기 (a)의 유전자는 하기 표 1에서 선택될 수 있다.In one specific example, the gene of (a) can be selected from Table 1 below.

NO.NO. 유전자gene 염색체 내 영역Region within a chromosome 11 MXRA8MXRA8 chr1-1289873-1290557chr1-1289873-1290557 22 PTPRUPTPRU chr1-29585903-29586139chr1-29585903-29586139 33 IL12RB2IL12RB2 chr1-67773330-67773579chr1-67773330-67773579 44 WNT3AWNT3A chr1-228225569-228225955chr1-228225569-228225955 55 KIF26BKIF26B chr1-245851386-245851485chr1-245851386-245851485 66 SLC30A3/DNAJC5GSLC30A3/DNAJC5G chr2-27487380-27487489chr2-27487380-27487489 77 ZFP36L2ZFP36L2 chr2-43451900-43452012chr2-43451900-43452012 88 ZIC4ZIC4 chr3-147113790-147113904chr3-147113790-147113904 99 CPLX1CPLX1 chr4-789186-789341chr4-789186-789341 1010 ZFYVE28ZFYVE28 chr4-2366608-2366816chr4-2366608-2366816 1111 CCNG2/CXCL13CCNG2/CXCL13 chr4-78114672-78114902chr4-78114672-78114902 1212 PALLDPALLD chr4-169799215-169799375chr4-169799215-169799375 1313 IRX4IRX4 chr5-1878570-1878706chr5-1878570-1878706 1414 C5orf38C5orf38 chr5-2755271-2755443chr5-2755271-2755443 1515 ISL1ISL1 chr5-50685660-50685990chr5-50685660-50685990 1616 LHFPL2LHFPL2 chr5-77805683-77805908chr5-77805683-77805908 1717 GPRIN1GPRIN1 chr5-176023779-176024240chr5-176023779-176024240 1818 LOC100506207/TFAP2ALOC100506207/TFAP2A chr6-10385242-10385390chr6-10385242-10385390 1919 GFOD1/SIRT5GFOD1/SIRT5 chr6-13561982-13562261chr6-13561982-13562261 2020 ETV7ETV7 chr6-36355849-36356153chr6-36355849-36356153 2121 FOXP4FOXP4 chr6-41528771-41528923chr6-41528771-41528923 2222 SMOC2SMOC2 chr6-168841499-168842200chr6-168841499-168842200 2323 GNA12/CARD11GNA12/CARD11 chr7-2903209-2903361chr7-2903209-2903361 2424 AP5Z1AP5Z1 chr7-4832070-4832235chr7-4832070-4832235 2525 ZNF273/ZNF117ZNF273/ZNF117 chr7-64408037-64408223chr7-64408037-64408223 2626 CALN1CALN1 chr7-71801254-71801414chr7-71801254-71801414 2727 SND1SND1 chr7-127671835-127671950chr7-127671835-127671950 2828 HTR5A/INSIG1HTR5A/INSIG1 chr7-154997216-154998032chr7-154997216-154998032 2929 MNX1MNX1 chr7-156798050-156798286chr7-156798050-156798286 3030 EBF2/PPP2R2AEBF2/PPP2R2A chr8-25907653-25907808chr8-25907653-25907808 3131 FAM110BFAM110B chr8-59058025-59058340chr8-59058025-59058340 3232 OSR2/VPS13BOSR2/VPS13B chr8-99986018-99986125chr8-99986018-99986125 3333 NAPRTNAPRT chr8-144659885-144660773chr8-144659885-144660773 3434 GLIS3GLIS3 chr9-4299929-4300086chr9-4299929-4300086 3535 SLC24A2/MLLT3SLC24A2/MLLT3 chr9-19934446-19934554chr9-19934446-19934554 3636 CRB2CRB2 chr9-126135490-126135634chr9-126135490-126135634 3737 RABL6RABL6 chr9-139715867-139715996chr9-139715867-139715996 3838 TACC2TACC2 chr10-123923101-123923437chr10-123923101-123923437 3939 CPXM2/CHST15CPXM2/CHST15 chr10-125751219-125751345chr10-125751219-125751345 4040 CLMPCLMP chr11-123066597-123066744chr11-123066597-123066744 4141 KRT7/KRT81KRT7/KRT81 chr12-52652331-52652515chr12-52652331-52652515 4242 HOXC4HOXC4 chr12-54447318-54447601chr12-54447318-54447601 4343 LOC100287944LOC100287944 chr12-106988280-106988380chr12-106988280-106988380 4444 LINC00403LINC00403 chr13-112707765-112707873chr13-112707765-112707873 4545 LINC00403LINC00403 chr13-112711235-112711462chr13-112711235-112711462 4646 LINC00925LINC00925 chr15-89921078-89921217chr15-89921078-89921217 4747 NR2F2-AS1NR2F2-AS1 chr15-96866980-96867257chr15-96866980-96867257 4848 MGRN1MGRN1 chr16-4714056-4714161chr16-4714056-4714161 4949 RBFOX1RBFOX1 chr16-6069329-6069430chr16-6069329-6069430 5050 PLEKHH3PLEKHH3 chr17-40822053-40822300chr17-40822053-40822300 5151 EMILIN2EMILIN2 chr18-2905913-2906068chr18-2905913-2906068 5252 ST8SIA3ST8SIA3 chr18-55020377-55020654chr18-55020377-55020654 5353 ST8SIA3/ONECUT2ST8SIA3/ONECUT2 chr18-55094946-55095079chr18-55094946-55095079 5454 ST8SIA3/ONECUT2ST8SIA3/ONECUT2 chr18-55096004-55096154chr18-55096004-55096154 5555 ST8SIA3/ONECUT2ST8SIA3/ONECUT2 chr18-55097474-55097592chr18-55097474-55097592 5656 ONECUT2ONECUT2 chr18-55101950-55102109chr18-55101950-55102109 5757 NFATC1/CTDP1NFATC1/CTDP1 chr18-77397555-77397677chr18-77397555-77397677 5858 PARD6GPARD6G chr18-77917687-77917849chr18-77917687-77917849 5959 ZNF563ZNF563 chr19-12444251-12444522chr19-12444251-12444522 6060 MED29/ZFP36MED29/ZFP36 chr19-39894639-39894823chr19-39894639-39894823 6161 DYRK1BDYRK1B chr19-40324031-40324746chr19-40324031-40324746 6262 PPP1R13LPPP1R13L chr19-45888913-45889303chr19-45888913-45889303 6363 CYP24A1CYP24A1 chr20-52789641-52789825chr20-52789641-52789825 6464 CYP24A1CYP24A1 chr20-52790137-52790317chr20-52790137-52790317 6565 C21orf2/TRPM2C21orf2/TRPM2 chr21-45770053-45770203chr21-45770053-45770203 6666 YBEYYBEY chr21-47717820-47717930chr21-47717820-47717930 6767 MAPK8IP2MAPK8IP2 chr22-51042493-51042765chr22-51042493-51042765

일 구체예에 있어서, 상기 (b)의 유전자는 하기 표 2에서 선택될 수 있다.In one specific example, the gene of (b) can be selected from Table 2 below.

NO.NO. 유전자gene 염색체 내 영역Region within a chromosome 11 LCKLCK chr1-32742005-32742140chr1-32742005-32742140 22 FCRL5FCRL5 chr1-157492943-157493093chr1-157492943-157493093 33 RHOU/RAB4ARHOU/RAB4A chr1-229374459-229374735chr1-229374459-229374735 44 GREM2/RGS7GREM2/RGS7 chr1-240817303-240817482chr1-240817303-240817482 55 DCDC2C/LINC01304DCDC2C/LINC01304 chr2-3990335-3990440chr2-3990335-3990440 66 RNU6-81PRNU6-81P chr2-132300645-132300786chr2-132300645-132300786 77 CCDC108CCDC108 chr2-219892467-219892617chr2-219892467-219892617 88 LOC150935/MIR4786LOC150935/MIR4786 chr2-240752453-240752653chr2-240752453-240752653 99 AQP12BAQP12B chr2-241622219-241622396chr2-241622219-241622396 1010 AQP12AAQP12A chr2-241631133-241631283chr2-241631133-241631283 1111 LINC01237LINC01237 chr2-242841595-242841745chr2-242841595-242841745 1212 XCR1XCR1 chr3-46069296-46069496chr3-46069296-46069496 1313 FGFR3FGFR3 chr4-1805102-1805218chr4-1805102-1805218 1414 NDST4/MIR1973NDST4/MIR1973 chr4-117218386-117218586chr4-117218386-117218586 1515 TPPPTPPP chr5-669788-669965chr5-669788-669965 1616 HNRNPH1/C5orf60HNRNPH1/C5orf60 chr5-179059331-179059604chr5-179059331-179059604 1717 RASGEF1CRASGEF1C chr5-179528232-179528389chr5-179528232-179528389 1818 LINC01012/LOC100131289LINC01012/LOC100131289 chr6-27706844-27707048chr6-27706844-27707048 1919 HLA-DPA1HLA-DPA1 chr6-33032739-33032881chr6-33032739-33032881 2020 HLA-DPA1HLA-DPA1 chr6-33035235-33035380chr6-33035235-33035380 2121 HLA-DPA1HLA-DPA1 chr6-33036446-33036546chr6-33036446-33036546 2222 HLA-DPA1HLA-DPA1 chr6-33037577-33037741chr6-33037577-33037741 2323 SMOC2/THBS2SMOC2/THBS2 chr6-169539688-169539949chr6-169539688-169539949 2424 ADAP1ADAP1 chr7-987360-987484chr7-987360-987484 2525 ELFN1ELFN1 chr7-1786348-1786448chr7-1786348-1786448 2626 MAD1L1MAD1L1 chr7-1891369-1891547chr7-1891369-1891547 2727 LYPD2LYPD2 chr8-143834030-143834584chr8-143834030-143834584 2828 MAPK15MAPK15 chr8-144801594-144801758chr8-144801594-144801758 2929 PUF60PUF60 chr8-144906207-144906369chr8-144906207-144906369 3030 LINGO2/LINC01242LINGO2/LINC01242 chr9-29826505-29826655chr9-29826505-29826655 3131 APBA1APBA1 chr9-72131795-72131950chr9-72131795-72131950 3232 NCS1NCS1 chr9-132955955-132956099chr9-132955955-132956099 3333 FIBCD1FIBCD1 chr9-133779363-133779564chr9-133779363-133779564 3434 FIBCD1FIBCD1 chr9-133779750-133779963chr9-133779750-133779963 3535 FIBCD1FIBCD1 chr9-133799133-133799306chr9-133799133-133799306 3636 RXRA/COL5A1RXRA/COL5A1 chr9-137356284-137356449chr9-137356284-137356449 3737 KCNT1KCNT1 chr9-138669335-138669459chr9-138669335-138669459 3838 GPSM1GPSM1 chr9-139230380-139230588chr9-139230380-139230588 3939 PFKFB3PFKFB3 chr10-6264447-6264547chr10-6264447-6264547 4040 ATRNL1ATRNL1 chr10-117668266-117668728chr10-117668266-117668728 4141 FRG2B/DUX4L3FRG2B/DUX4L3 chr10-135453991-135454183chr10-135453991-135454183 4242 PKP3PKP3 chr11-394531-394708chr11-394531-394708 4343 MUC5BMUC5B chr11-1263116-1263266chr11-1263116-1263266 4444 LMO1LMO1 chr11-8248450-8248604chr11-8248450-8248604 4545 LOC441601LOC441601 chr11-50257274-50257872chr11-50257274-50257872 4646 APOA5APOA5 chr11-116661162-116661443chr11-116661162-116661443 4747 TRIM29TRIM29 chr11-119992252-119992572chr11-119992252-119992572 4848 B4GALNT3B4GALNT3 chr12-670471-670650chr12-670471-670650 4949 CACNA2D4LRTM2CACNA2D4LRTM2 chr12-1936192-1936486chr12-1936192-1936486 5050 TSPAN9/PRMT8TSPAN9/PRMT8 chr12-3476351-3476555chr12-3476351-3476555 5151 VAMP1/MRPL51VAMP1/MRPL51 chr12-6586920-6587068chr12-6586920-6587068 5252 GNB3GNB3 chr12-6949911-6950160chr12-6949911-6950160 5353 LRIG3/SLC16A7LRIG3/SLC16A7 chr12-59924804-59925230chr12-59924804-59925230 5454 CABP1CABP1 chr12-121104906-121105088chr12-121104906-121105088 5555 TMEM132DTMEM132D chr12-130334337-130334502chr12-130334337-130334502 5656 LINC01257LINC01257 chr12-131648973-131649117chr12-131648973-131649117 5757 LOC101928416/FBRSL1LOC101928416/FBRSL1 chr12-133009259-133009447chr12-133009259-133009447 5858 LINC00572LINC00572 chr13-30498612-30498734chr13-30498612-30498734 5959 MCF2LMCF2L chr13-113732740-113732871chr13-113732740-113732871 6060 MCF2LMCF2L chr13-113732999-113733179chr13-113732999-113733179 6161 GRTP1/ADPRHL1GRTP1/ADPRHL1 chr13-114064805-114065135chr13-114064805-114065135 6262 EAPP/SNX6EAPP/SNX6 chr14-35021946-35022142chr14-35021946-35022142 6363 ELK2AP/KIAA0125ELK2AP/KIAA0125 chr14-106145858-106146008chr14-106145858-106146008 6464 ADAM6/LINC00226ADAM6/LINC00226 chr14-106539847-106539997chr14-106539847-106539997 6565 LINC00226/LINC00221LINC00226/LINC00221 chr14-106857323-106857476chr14-106857323-106857476 6666 C15orf48C15orf48 chr15-45723131-45723303chr15-45723131-45723303 6767 SV2BSV2B chr15-91840105-91840252chr15-91840105-91840252 6868 LOC101927332/FAM169BLOC101927332/FAM169B chr15-98647977-98648112chr15-98647977-98648112 6969 SBK1SBK1 chr16-28332430-28332589chr16-28332430-28332589 7070 RRN3P2/SNX29P2RRN3P2/SNX29P2 chr16-29150987-29151139chr16-29150987-29151139 7171 ARHGEF15ARHGEF15 chr17-8219200-8219419chr17-8219200-8219419 7272 NFE2L1NFE2L1 chr17-46131641-46131830chr17-46131641-46131830 7373 SEC14L1/SEPT9SEC14L1/SEPT9 chr17-75276276-75276449chr17-75276276-75276449 7474 B3GNTL1/METRNLB3GNTL1/METRNL chr17-81025581-81025810chr17-81025581-81025810 7575 AQP4-AS1AQP4-AS1 chr18-24446445-24446635chr18-24446445-24446635 7676 TSHZ1TSHZ1 chr18-72924982-72925238chr18-72924982-72925238 7777 GALR1/LINC01029GALR1/LINC01029 chr18-75402175-75402375chr18-75402175-75402375 7878 LINC01029, SALL3LINC01029, SALL3 chr18-76690593-76690779chr18-76690593-76690779 7979 GRIN3BGRIN3B chr19-1003445-1003566chr19-1003445-1003566 8080 SMIM24/DOHHSMIM24/DOHH chr19-3482825-3482999chr19-3482825-3482999 8181 C19orf67C19orf67 chr19-14196529-14196706chr19-14196529-14196706 8282 TMC4TMC4 chr19-54668914-54669091chr19-54668914-54669091 8383 LILRB3LILRB3 chr19-54724045-54724314chr19-54724045-54724314 8484 SIRPG/LOC100289473SIRPG/LOC100289473 chr20-1716350-1716478chr20-1716350-1716478 8585 FOXS1FOXS1 chr20-30432958-30433165chr20-30432958-30433165 8686 HELZ2HELZ2 chr20-62194775-62194880chr20-62194775-62194880 8787 LOC101927797/LINC00320LOC101927797/LINC00320 chr21-21272245-21272489chr21-21272245-21272489 8888 LINC00111LINC00111 chr21-43109581-43109724chr21-43109581-43109724 8989 TFF2TFF2 chr21-43771286-43771456chr21-43771286-43771456 9090 ZDHHC8P1ZDHHC8P1 chr22-23732095-23732326chr22-23732095-23732326 9191 LOC284930LOC284930 chr22-48027673-48027833chr22-48027673-48027833

본 명세서에서, "유전자 A/유전자 B"는 두 유전자 사이에 존재하는 영역을 의미하며, 구체적으로 두 유전자의 영역을 제외한 두 유전자 사이의 영역을 의미한다. 즉, "유전자 A/유전자 B"는 유전자 A의 영역 및 유전자 B의 영역은 포함하지 않고, 유전자 A와 유전자 B의 사이 영역을 포함한다.In this specification, "gene A/gene B" means a region existing between two genes, and specifically means a region between two genes excluding the regions of the two genes. That is, "gene A/gene B" does not include the region of gene A and the region of gene B, but includes the region between gene A and gene B.

본 명세서에서 상기 유전자의 CpG 영역을 갖는 단편은, 해당 유전자의 CpG 영역을 갖는 단편들 중 해당 유전자를 대표할 수 있도록 해당 유전자에 대해 선택성 및 특이성을 갖는 단편을 의미할 수 있다. 따라서, 임의의 유전자의 CpG 영역을 갖는 단편의 메틸화 수준을 측정하여 해당 유전자의 메틸화 수준을 비교할 수 있고 이에 따라 에드워드 증후군을 진단할 수 있다.In this specification, a fragment having a CpG region of the gene may mean a fragment having selectivity and specificity for the gene so as to represent the gene among fragments having the CpG region of the gene. Accordingly, by measuring the methylation level of a fragment having a CpG region of any gene, the methylation level of the gene can be compared, and Edwards syndrome can be diagnosed accordingly.

또한 임의의 유전자의 CpG 영역의 위치는 당업계에 알려져 있으므로, 당업자는 본 발명의 목적하는 효과를 구현하기 위하여 유전자 내 CpG 영역을 갖는 단편을 적절히 선택하거나, 당업계에 임의의 유전자를 대표하는 것으로 알려진 CpG 영역을 갖는 단편을 이용할 수 있다. 또한, 이러한 단편의 메틸화 수준을 측정하는 제제를 구입하여 목적하는 유전자의 메틸화 수준을 비교할 수 있다.In addition, since the location of the CpG region of any gene is known in the art, those skilled in the art can appropriately select a fragment having a CpG region in the gene to realize the desired effect of the present invention, or can utilize a fragment having a CpG region known in the art to represent any gene. In addition, a preparation for measuring the methylation level of such a fragment can be purchased to compare the methylation level of the desired gene.

일 구체예에 있어서, 상기 유전자 또는 상기 유전자의 CpG 영역을 갖는 단편은, 모체, 정상 태아 및 에드워드증후군 태아 순으로 CpG 영역의 메틸화 수준이 증가하거나 또는 감소하는 후성학적 특성이 연속적으로 2개 이상 존재하는 부위로서, 상기 후성학적 특성이 유지되는 부위를 가지는 유전자 또는 이의 단편일 수 있다.In one specific example, the gene or a fragment having a CpG region of the gene may be a gene or a fragment thereof having a region where two or more epigenetic characteristics in which the methylation level of the CpG region increases or decreases sequentially in the order of the mother, a normal fetus, and an Edwards syndrome fetus exist, and where the epigenetic characteristics are maintained.

본 명세서에서 용어 "수준 측정"은 에드워드 증후군을 진단하기 위하여 생물학적 시료에서 에드워드 증후군에 대한 마커의 메틸화 정도를 측정하는 것을 포함할 수 있다. 뿐만이 아니라, 상기 마커의 발현 또는 활성의 정도를 측정하는 것을 포함할 수 있다.The term "level measurement" herein may include measuring the degree of methylation of a marker for Edwards syndrome in a biological sample to diagnose Edwards syndrome. In addition, it may include measuring the degree of expression or activity of said marker.

보다 구체적으로, 본 명세서에서 용어 "CpG 영역의 메틸화 수준 측정"은 상기 유전자의 CpG 영역의 메틸화 수준을 측정하는 것을 의미할 수 있으며, Methyl capture sequencing(MC-seq), 중합효소연쇄반응(Polymerase Chain Reaction, PCR), 메틸화 특이 PCR(methylation specific PCR), 실시간 메틸화 특이 PCR(real time methylation specific PCR), 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 메틸 PNA-PCR, 정량 PCR, DNA 칩, 파이로시퀀싱 및 바이설파이트시퀀싱으로 이루어진 군으로부터 선택되는 어느 하나 이상에 의해 수행될 수 있으나, 이에 제한되는 것은 아니다.More specifically, the term "measuring the methylation level of a CpG region" in the present specification may mean measuring the methylation level of a CpG region of the gene, and may be performed by at least one selected from the group consisting of, but not limited to, Methyl capture sequencing (MC-seq), Polymerase Chain Reaction (PCR), methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA-specific binding protein, methyl PNA-PCR, quantitative PCR, DNA chip, pyrosequencing, and bisulfite sequencing.

또한, 상기 CpG 영역의 메틸화 수준 측정은 유전체 수준에서 측정된 것일 수 있다.Additionally, the measurement of the methylation level of the above CpG region may be measured at the genome level.

본 명세서에서 용어 "마커(marker)" 또는 "바이오마커(biomarker)"란 면역 관련 질병을 가지는 개체와 면역 관련 질병을 가지지 않은 개체를 구분하여 평가할 수 있는 물질을 의미하며, 면역 관련 질병을 가지지 않은 개체에 비하여 면역 관련 질병을 가지는 개체에서는 증가 또는 감소를 보이는 폴리펩타이드, 핵산(예컨대, mRNA 등), 지질, 당지질, 당단백질, 당(단당류, 이당류, 올리고당류 등), 대사체 등과 같은 유기 생체 분자 등을 포함할 수 있다.The term "marker" or "biomarker" as used herein refers to a substance that can be evaluated to distinguish between individuals having an immune-related disease and individuals not having an immune-related disease, and may include organic biomolecules such as polypeptides, nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), metabolites, etc., which show an increase or decrease in individuals having an immune-related disease compared to individuals not having the immune-related disease.

본 명세서에서 용어 "진단"은 특정 질병 또는 상태에 대한 객체의 감수성을 판정하는 것, 객체가 특정 질병 또는 상태를 가지고 있는지 여부를 판정하는 것, 특정 질병 또는 상태인 객체의 예후를 판정하는 것, 또는 이를 모니터링하는 것을 지칭할 수 있다.The term "diagnosis" herein may refer to determining a subject's susceptibility to a particular disease or condition, determining whether a subject has a particular disease or condition, determining a prognosis for a subject having a particular disease or condition, or monitoring the same.

본 명세서에서 용어 "에드워드 증후군"은 18번 염색체가 3개 존재하는 염색체 이상으로 유발되는 유전 질환(삼염색체성 유전질환)을 의미한다. The term "Edwards syndrome" in this specification refers to a genetic disorder (trisomy genetic disorder) caused by a chromosomal abnormality in which there are three copies of chromosome 18.

상기 에드워드 증후군의 병태는 선천적 외형 기형, 외형상 작고 좁은 머리, 후두골 돌출, 소구증, 구순열 및 구개열, 소하악증, 짧은 흉골, 작은 골반, 특징적인 손발 모양(집게손가락이 가운뎃손가락 위에, 새끼손가락이 넷째 손가락 위에 덮인 손 모양), 오목발바닥이면서 뒤꿈치가 두드러진 모양(rocker-bottom feet), 짧고 뒤로 굴곡된 첫째 발가락, 발달 지연, 정신 지체, 심한 정신 지체, 심장 기형(심실 중격 결손, 동맥관개존증), 신장 기형(마제신), 횡경막 탈장, 기관 식도루, 호흡 장애, 근육 약화 등일 수 있다.The above Edwards syndrome pathologies may include congenital external malformations, externally small and narrow head, occipital prominence, microstomia, cleft lip and palate, micrognathia, short sternum, small pelvis, characteristic hand and foot shapes (index finger over middle finger and little finger over fourth finger), rocker-bottom feet, short and backward-bent first toes, developmental delay, mental retardation, severe mental retardation, heart malformations (ventricular septal defect, patent ductus arteriosus), renal malformations (horsehair kidney), diaphragmatic hernia, tracheoesophageal fistula, respiratory distress, and muscle weakness.

일 구체예에 있어서, 상기 유전자의 CpG 영역의 메틸화 수준을 측정하는 제제는, 바이설파이트(bisulfite) 또는 이의 염, 메틸화 민감성 제한효소, 상기 유전자의 CpG 부위를 포함하는 서열에 특이적으로 결합하는 프라이머, 상기 유전자의 메틸화된 CpG 부위를 포함하는 서열에 혼성화할 수 있는 프로브, 메틸화된 CpG 결합 도메인, 또는 메틸사이토신에 특이적으로 결합하는 항체 또는 압타머를 포함할 수 있다.In one specific example, the agent for measuring the methylation level of the CpG region of the gene may include bisulfite or a salt thereof, a methylation-sensitive restriction enzyme, a primer that specifically binds to a sequence including a CpG site of the gene, a probe that can hybridize to a sequence including a methylated CpG site of the gene, a methylated CpG binding domain, or an antibody or aptamer that specifically binds to methylcytosine.

다른 양상은 상기 조성물을 포함하는, 에드워드 증후군 진단용 키트를 제공한다.Another aspect provides a kit for diagnosing Edwards syndrome, comprising the composition.

상기 에드워드 증후군은 전술한 바와 같다.The above Edwards syndrome is as described above.

일 구체예에 있어서, 상기 키트는 (i) 상기한 바와 같은 에드워드 증후군 진단에 특이적 유전자 또는 이의 단편에 혼성화될 수 있는 다수의 프라이머, 프로브 또는 안티센스 뉴클레오티드; (ii) 상기 유전자 또는 이의 단편에 혼성화될 수 있는 다수의 프라이머, 프로브 또는 안티센스 뉴클레오티드 및 대상의 생물학적 샘플(예컨대, 생물학적 시료로부터 분리된 핵산)을 함유하기에 적합한 용기; (iii) (ii)의 혼성화를 검출하기 위한 수단; 및/또는 선택적으로 (iv) 사용 설명서 및 키트 결과의 해석을 포함할 수 있다.In one embodiment, the kit may comprise (i) a plurality of primers, probes or antisense nucleotides capable of hybridizing to a gene specific for diagnosing Edwards syndrome, or a fragment thereof, as described above; (ii) a container suitable for containing a plurality of primers, probes or antisense nucleotides capable of hybridizing to the gene or a fragment thereof and a biological sample of the subject (e.g., a nucleic acid isolated from the biological sample); (iii) means for detecting the hybridization of (ii); and/or optionally (iv) instructions for use and interpretation of the kit results.

상기 키트는 또한 별도의 용기에 패키징된 혼성화 용액을 포함한 다른 성분들을 함유할 수 있다. 상기 혼성화 용액에서 상기 유전자의 핵산과 다수의 프라이머, 프로브 또는 안티센스 뉴클레오티드가 혼성화될 수 있다.The above kit may also contain other components, including a hybridization solution packaged in a separate container, in which the nucleic acid of the gene can be hybridized with a plurality of primers, probes or antisense nucleotides.

구체적으로, 상기 키트는 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성성분 조성물, 용액 또는 장치로 구성될 수 있다. 예를 들어, 상기 키트는 RT-PCR(Reverse transcription polymerase chain reaction) 키트, DNA 칩 키트일 수 있다. 상기 키트는 상기 제제 이외에도 중합효소 아가로스, 전기영동에 필요한 완충용액 등이 추가로 포함될 수 있다.Specifically, the kit may be composed of one or more types of other component compositions, solutions or devices suitable for the analysis method. For example, the kit may be an RT-PCR (Reverse transcription polymerase chain reaction) kit, a DNA chip kit. In addition to the above formulation, the kit may additionally include a polymerase agarose, a buffer solution required for electrophoresis, etc.

일 구체예에 있어서, 상기 키트는, 상기 CpG 영역을 갖는 유전자 또는 CpG 영역을 갖는 이의 단편과 혼성화할 수 있는 프로브가 고정되어 있는 진단용 핵산 칩(CHIP) 또는 유전자 패널을 포함할 수 있다.In one specific example, the kit may include a diagnostic nucleic acid chip (CHIP) or gene panel having a probe immobilized thereon, which is capable of hybridizing with a gene having the CpG region or a fragment thereof having the CpG region.

일 구체예에 있어서, 상기 키트는 상기 유전자 또는 이의 단편의 CpG 영역의 메틸화 수준을 측정하기 위한 제제, 장치, 및 알고리즘이 내장된 컴퓨터를 포함할 수 있고, 상기 알고리즘을 통해 상기 마커의 수준 측정 결과를 에드워드 증후군 진단과 연관시키는 것인, 키트에 관한 것일 수 있다.In one specific example, the kit may include a computer having a built-in agent, device, and algorithm for measuring the methylation level of a CpG region of the gene or a fragment thereof, and may relate to a kit that associates the result of measuring the level of the marker with a diagnosis of Edwards syndrome through the algorithm.

또 다른 양상은 대상체로부터 분리된 태반시료에서 CpG 영역을 갖는 유전자의 CpG 영역의 메틸화 수준을 측정하는 단계; 및 상기 단계에서 측정된 메틸화 수준을 모체 혈액 및 정상 태반 내 동일 유전자의 CpG 영역의 메틸화 수준과 비교하는 단계를 포함하는, 에드워드증후군의 진단을 위한 정보를 제공하는 방법을 제공한다.Another aspect provides a method for providing information for diagnosing Edwards syndrome, comprising the steps of: measuring a methylation level of a CpG region of a gene having a CpG region in a placental sample isolated from a subject; and comparing the methylation level measured in the step with the methylation level of the CpG region of the same gene in maternal blood and a normal placenta.

또 다른 양상은 대상체로부터 분리된 태반시료에서 CpG 영역을 갖는 유전자의 CpG 영역의 메틸화 수준을 측정하는 단계; 및 상기 단계에서 측정된 메틸화 수준을 모체 혈액 및 정상 태반 내 동일 유전자의 CpG 영역의 메틸화 수준과 비교하는 단계를 포함하는, 에드워드증후군의 진단 방법을 제공한다.Another aspect provides a method for diagnosing Edwards syndrome, comprising the steps of: measuring a methylation level of a CpG region of a gene having a CpG region in a placental sample isolated from a subject; and comparing the methylation level measured in the step with the methylation level of the CpG region of the same gene in maternal blood and a normal placenta.

일 구체예에 있어서, 상기 태반은 출산 후 배출되는 태반이 아닌, 초기산전검사인 CVS(Chorionic Villi Sampling)에 사용되는 융모막 검체를 의미할 수 있다.In one specific example, the placenta may refer to a chorionic villi sample used in CVS (Chorionic Villi Sampling), which is an early prenatal test, rather than a placenta discharged after birth.

상기 태반, CpG 영역, 수준 측정, CpG 영역의 메틸화 수준 측정, 진단 및 에드워드 증후군은 전술한 바와 같다.The placenta, CpG region, level measurement, methylation level measurement of CpG region, diagnosis and Edwards syndrome are as described above.

일 구체예에 있어서, 상기 메틸화 수준을 측정하는 단계는 PCR, 메틸화 특이적 PCR(methylation-specific polymerase chain reaction), 실시간 메틸화 특이적 PCR(real time methylation-specific polymerase chain reaction), MethyLight PCR, MehtyLight digital PCR, EpiTYPER, 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 메틸 PNA-PCR, 정량 PCR, DNA 칩, 파이로시퀀싱, 바이설파이트 시퀀싱, 서던블롯, RLGS(Restriction landmark genomic scanning), MS-SnuPE(Methylation-sensitive single-nucleotide primer extension), CpG 마이크로어레이, COBRA(Combined bisulfite-restriction analysis), MIRA(methylated-CpG island recovery assay), 질량 스펙트럼법, 및 메틸화된 CpG 결합 도메인 또는 항-메틸사이토신 항체를 이용한 면역침강법으로 이루어진 군으로부터 선택되는 어느 하나 이상에 의해 수행되는 것일 수 있다.In one specific example, the step of measuring the methylation level may be performed by at least one selected from the group consisting of PCR, methylation-specific PCR, real time methylation-specific PCR, MethyLight PCR, MehtyLight digital PCR, EpiTYPER, PCR using methylated DNA-specific binding protein, methyl PNA-PCR, quantitative PCR, DNA chip, pyrosequencing, bisulfite sequencing, Southern blot, RLGS (Restriction landmark genomic scanning), MS-SnuPE (Methylation-sensitive single-nucleotide primer extension), CpG microarray, COBRA (Combined bisulfite-restriction analysis), MIRA (methylated-CpG island recovery assay), mass spectrometry, and immunoprecipitation using a methylated CpG binding domain or an anti-methylcytosine antibody.

일 구체예에 있어서, 상기 유전자는 하기 (a) 및 (b)로 이루어진 군에서 선택되는 하나 이상의 유전자를 포함하는 것일 수 있다:In one specific example, the gene may comprise one or more genes selected from the group consisting of (a) and (b):

(a) MXRA8, PTPRU, IL12RB2, WNT3A, KIF26B, SLC30A3/DNAJC5G, ZFP36L2, ZIC4, CPLX1, ZFYVE28, CCNG2/CXCL13, PALLD, IRX4, C5orf38, ISL1, LHFPL2, GPRIN1, LOC100506207/TFAP2A, GFOD1/SIRT5, ETV7, FOXP4, SMOC2, GNA12/CARD11, AP5Z1, ZNF273/ZNF117, CALN1, SND1, HTR5A/INSIG1, MNX1, EBF2/PPP2R2A, FAM110B, OSR2/VPS13B, NAPRT, GLIS3, SLC24A2/MLLT3, CRB2, RABL6, TACC2, CPXM2/CHST15, CLMP, KRT7/KRT81, HOXC4, LOC100287944, LINC00403, LINC00925, NR2F2-AS1, MGRN1, RBFOX1, PLEKHH3, EMILIN2, ST8SIA3, ST8SIA3/ONECUT2, ONECUT2, NFATC1/CTDP1, PARD6G, ZNF563, MED29/ZFP36, DYRK1B, PPP1R13L, CYP24A1, C21orf2/TRPM2, YBEY, MAPK8IP2 및 이들의 CpG 영역을 갖는 단편; 및 (a) MXRA8, PTPRU, IL12RB2, WNT3A, KIF26B, SLC30A3/DNAJC5G, ZFP36L2, ZIC4, CPLX1, ZFYVE28, CCNG2/CXCL13, PALLD, IRX4, C5orf38, ISL1, LHFPL2, GPRIN1, LOC100506207/TFAP2A, GFOD1/SIRT5, ETV7, FOXP4, SMOC2, GNA12/CARD11, AP5Z1, ZNF273/ZNF117, CALN1, SND1, HTR5A/INSIG1, MNX1, EBF2/PPP2R2A, FAM110B, OSR2/VPS13B, NAPRT, GLIS3, SLC24A2/MLLT3, CRB2, RABL6, TACC2, CPXM2/CHST15, CLMP, KRT7/KRT81, HOXC4, LOC100287944, LINC00403, LINC00925, NR2F2-AS1, MGRN1, RBFOX1, PLEKHH3, EMILIN2, ST8SIA3, ST8SIA3/ONECUT2, ONECUT2, NFATC1/CTDP1, PARD6G, ZNF563, MED29/ZFP36, DYRK1B, PPP1R13L, CYP24A1, C21orf2/TRPM2, YBEY, MAPK8IP2 and fragments having their CpG regions; and

(b) LCK, FCRL5, RHOU/RAB4A, GREM2/RGS7, DCDC2C/LINC01304, RNU6-81P, CCDC108, LOC150935/MIR4786, AQP12B, AQP12A, LINC01237, XCR1, FGFR3, NDST4/MIR1973, TPPP, HNRNPH1/C5orf60, RASGEF1C, LINC01012/LOC100131289, HLA-DPA1, SMOC2/THBS2, ADAP1, ELFN1, MAD1L1, LYPD2, MAPK15, PUF60, LINGO2/LINC01242, APBA1, NCS1, FIBCD1, RXRA/COL5A1, KCNT1, GPSM1, PFKFB3, ATRNL1, FRG2B/DUX4L3, PKP3, MUC5B, LMO1, LOC441601, APOA5, TRIM29, B4GALNT3, CACNA2D4/LRTM2, TSPAN9/PRMT8, VAMP1/MRPL51, GNB3, LRIG3/SLC16A7, CABP1, TMEM132D, LINC01257, LOC101928416/FBRSL1, LINC00572, MCF2L, GRTP1/ADPRHL1, EAPP/SNX6, ELK2AP/KIAA0125, ADAM6/LINC00226, , LINC00226/LINC00221, C15orf48, SV2B, LOC101927332/FAM169B, SBK1, RRN3P2/SNX29P2, ARHGEF15, NFE2L1, SEC14L1/SEPT9, B3GNTL1/METRNL, AQP4-AS1, TSHZ1, GALR1/LINC01029, LINC01029/SALL3, GRIN3B, SMIM24/DOHH, C19orf67, TMC4, LILRB3, SIRPG/LOC100289473, FOXS1, HELZ2, LOC101927797/LINC00320, LINC00111, TFF2, ZDHHC8P1, LOC284930 및 이들의 CpG 영역을 갖는 단편. (b) LCK, FCRL5, RHOU/RAB4A, GREM2/RGS7, DCDC2C/LINC01304, RNU6-81P, CCDC108, LOC150935/MIR4786, AQP12B, AQP12A, LINC01237, XCR1, FGFR3, NDST4/MIR1973, TPPP, HNRNPH1/C5orf60, RASGEF1C, LINC01012/LOC100131289, HLA-DPA1, SMOC2/THBS2, ADAP1, ELFN1, MAD1L1, LYPD2, MAPK15, PUF60, LINGO2/LINC01242, APBA1, NCS1, FIBCD1, RXRA/COL5A1, KCNT1, GPSM1, PFKFB3, ATRNL1, FRG2B/DUX4L3, PKP3, MUC5B, LMO1, LOC441601, APOA5, TRIM29, B4GALNT3, CACNA2D4/LRTM2, TSPAN9/PRMT8, VAMP1/MRPL51, GNB3, LRIG3/SLC16A7, CABP1, TMEM132D, LINC01257, LOC101928416/FBRSL1, LINC00572, MCF2L, GRTP1/ADPRHL1, EAPP/SNX6, ELK2AP/KIAA0125, ADAM6/LINC00226, , LINC00226/LINC00221, C15orf48, SV2B, LOC101927332/FAM169B, SBK1, RRN3P2/SNX29P2, ARHGEF15, NFE2L1, SEC14L1/SEPT9, B3GNTL1/METRNL, AQP4-AS1, TSHZ1, GALR1/LINC01029, LINC01029/SALL3, GRIN3B, SMIM24/DOHH, C19orf67, TMC4, LILRB3, SIRPG/LOC100289473, FOXS1, HELZ2, LOC101927797/LINC00320, LINC00111, TFF2, ZDHHC8P1, LOC284930 and fragments having their CpG regions.

일 구체예에 있어서, 상기 (a)의 유전자는 상기 표 1에서 선택될 수 있다.In one specific example, the gene of (a) can be selected from Table 1.

일 구체예에 있어서, 상기 (b)의 유전자는 상기 표 2에서 선택될 수 있다.In one specific example, the gene of (b) can be selected from Table 2.

일 구체예에 있어서, 상기 에드워드증후군의 진단을 위한 정보를 제공하는 방법은 상기 (a)의 유전자의 메틸화 수준이 모체 혈액 및 정상 태반 내 동일 유전자의 CpG 영역의 메틸화 수준에 비해 증가된 경우, 에드워드증후군으로 판단하거나, 상기 (b)의 유전자의 메틸화 수준이 모체 혈액 및 정상 태반 내 동일 유전자의 CpG 영역의 메틸화 수준에 비해 감소된 경우, 에드워드 증후군으로 판단하는 단계를 더 포함하는 것일 수 있다.In one specific example, the method for providing information for diagnosing Edwards syndrome may further include a step of determining Edwards syndrome when the methylation level of the gene of (a) is increased compared to the methylation level of the CpG region of the same gene in maternal blood and normal placenta, or determining Edwards syndrome when the methylation level of the gene of (b) is decreased compared to the methylation level of the CpG region of the same gene in maternal blood and normal placenta.

본 명세서에서 용어 "유전자의 메틸화 수준 증가" 또는 "유전자의 CpG 영역의 메틸화 수준 증가"는 대조군에 비해 대상체의 시료 내 메틸화 수준이 측정 가능할 정도로 유의하게 증가된 것을 의미하며, 예를 들어 약 1.1배 이상, 예를 들어 1.1배 내지 2.5배, 1.1배, 1.2배, 1.3배, 1.4배, 1.5배, 1.6배, 1.7배, 1.8배, 1.9배, 또는 2배 이상 증가된 경우를 의미할 수 있다.The term “increased methylation level of a gene” or “increased methylation level of a CpG region of a gene” as used herein means that the methylation level in a sample of a subject is measurably and significantly increased compared to a control, and may mean, for example, an increase of about 1.1-fold or more, for example, 1.1-fold to 2.5-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold or more.

본 명세서에서 용어 "유전자의 메틸화 수준 감소" 또는 "유전자의 CpG 영역의 메틸화 수준 감소"는 대조군에 비해 대상체의 시료 내 메틸화 수준이 측정 가능할 정도로 유의하게 감소된 것을 의미하며, 예를 들어 약 0.9배 이하, 예를 들어 0.1배 내지 0.9배, 0.9배, 0.8배, 0.7배, 0.6배, 0.5배, 0.4배, 0.3배, 0.2배, 또는 0.1배 이하로 감소된 경우를 의미할 수 있다.The term "decreased methylation level of a gene" or "decreased methylation level of a CpG region of a gene" as used herein means a measurable and significant decrease in the methylation level in a sample of a subject compared to a control, for example, about 0.9-fold or less, for example, 0.1-fold to 0.9-fold, 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, or 0.1-fold or less.

일 구체예에 있어서, 상기 에드워드 증후군을 진단하는 방법은 다수의 패널(panel)을 이용하는 형태로 수행될 수 있다.In one specific example, the method for diagnosing Edwards syndrome can be performed using a plurality of panels.

일 양상에 따른 에드워드 증후군 진단용 조성물 또는 이의 진단을 위한 정보를 제공하는 방법은 환자에게서 변화하는 질환 특이적인 후성학적 바이오마커의 메틸화 수준을 측정 및 비교하여 기존 고가의 검사 기기 혹은 특수 알고리즘의 필요 없이 에드워드 증후군을 간단하고 효과적으로 진단할 수 있다.A composition for diagnosing Edwards syndrome according to one aspect or a method for providing information for diagnosing the same can simply and effectively diagnose Edwards syndrome without the need for expensive existing testing devices or special algorithms by measuring and comparing the methylation level of disease-specific epigenetic biomarkers that change in a patient.

도 1 및 도 2는 에드워드 증후군 진단용으로 선정된 에드워드 증후군 특이적 과메틸화 바이오마커의 수준을 히트맵(heat map)분석한 결과이다.Figures 1 and 2 show the results of a heat map analysis of the levels of Edwards syndrome-specific hypermethylation biomarkers selected for the diagnosis of Edwards syndrome.

도 3 및 도 4는 에드워드 증후군 진단용으로 선정된 에드워드 증후군 특이적 저메틸화 바이오마커의 수준을 히트맵(heat map)분석한 결과이다.Figures 3 and 4 show the results of a heat map analysis of the levels of Edwards syndrome-specific hypomethylated biomarkers selected for the diagnosis of Edwards syndrome.

이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다. 실시예들은 다양한 변환을 가할 수 있는 바, 실시예들은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 다양한 형태로 구현될 수 있다.Hereinafter, preferred embodiments are presented to help understand the present invention. However, the following embodiments are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following embodiments. The embodiments can be modified in various ways, and the embodiments are not limited to the embodiments disclosed below, but can be implemented in various forms.

본 발명의 명세서 및 청구범위에 사용된 용어 또는 단어는 통상적이거나 사전적인 의미로 한정 해석되지 아니하며, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다.Terms or words used in the specification and claims of the present invention are not to be construed as limited to their usual or dictionary meanings, and should be interpreted as meanings and concepts that conform to the technical idea of the present invention based on the principle that the inventor can appropriately define the concept of the term in order to explain his or her own invention in the best manner.

본 발명의 명세서 전체에 있어서, 어떤 부분이 어떤 구성 요소를 "포함" 한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성 요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.Throughout the specification of the present invention, when a part is said to "include" a certain component, this does not exclude other components, but rather means that other components may be included, unless otherwise specifically stated.

본 발명의 명세서 전체에 있어서, "A 및/또는 B"는, A 또는 B, 또는 A 및 B를 의미한다.Throughout the specification of the present invention, “A and/or B” means A or B, or A and B.

실시예 1. 혈액 유전체 추출Example 1. Blood genome extraction

모체 5례의 혈액 시료에서 유전체를 추출하였다. Genomes were extracted from blood samples from five maternal cases.

Qiagen사의 DNeasy Blood & Tissue mini kits를 사용하여 제조사 제공 방식에 따라 수행하였으며, 세부적인 방법은 다음과 같다. Axygen사의 1.5ml 튜브에 프로틴아제 K 20 μl, RNase 20 μl, 시료 200 μl, AL 버퍼 200 μl를 넣고 혼합한 후 56 ℃에서 20 분간 인큐베이션을 실시하였다. 이어서 100% 에탄올을 시료에 추가하고 혼합한 후, 혼합물을 DNeasy Mini 스핀 컬럼으로 이송하여 8000 rpm에서 1분 동안 원심 분리를 수행하였다. 정제를 위해 Buffer AW1 500 ml을 추가하여 원심 분리를 한 후, Buffer AW2 500 μl을 추가하여 또 다시 원심 분리를 하였다. DNeasy Mini 스핀 컬럼을 이용하여 정제된 시료를 회수하고, buffer AE 50 μl을 추가하여 DNA를 염기화한 후 회수하였다. 최종적으로, 정제 단계를 반복하여 총 volume이 100 μl이 되도록 작업을 완료하였다.DNeasy Blood & Tissue mini kits from Qiagen were used according to the manufacturer's instructions, and the detailed methods are as follows. 20 μl of proteinase K, 20 μl of RNase, 200 μl of sample, and 200 μl of AL buffer were added to a 1.5 ml tube from Axygen, mixed, and incubated at 56 °C for 20 minutes. Then, 100% ethanol was added to the sample, mixed, and the mixture was transferred to a DNeasy Mini spin column and centrifuged at 8,000 rpm for 1 minute. For purification, 500 ml of Buffer AW1 was added, centrifuged, and then 500 μl of Buffer AW2 was added, centrifuged again. The purified sample was recovered using the DNeasy Mini spin column, and 50 μl of buffer AE was added to base-convert the DNA, and then recovered. Finally, the purification steps were repeated to complete the work until the total volume became 100 μl.

실시예 2. 태반 유전체 추출Example 2. Extraction of placental genome

정상 태아 5례 및 에드워드 증후군 태아 5례의 태반 시료에서 유전체를 추출하였다.Genomes were extracted from placental samples from five normal fetuses and five Edwards syndrome fetuses.

조직을 1.5ml 마이크로 튜브 (Axygen)에 넣고, 3 mm의 텅스텐 카바이드 비드 및 20 μl의 프로틴아제 K, 200 μl의 ATL 버퍼를 추가한 후 Tissuelyzer를 25 Hz에서 3 분간 작동시켰다. 이후 56℃에서 20분간 인큐베이션을 실시하였다. 이후 과정은 Qiagen사의 DNeasy Blood & Tissue mini kits를 사용하여 제조사 제공 방식에 따라 수행하였으며, 세부적인 방법은 다음과 같다. RNase 20 μl 및 AL 버퍼 200 μl을 추가한 후, 튜브를 완전히 혼합한다. 시료에 100% 에탄올을 첨가하고, 시료가 완전히 혼합되도록 섞는다. 혼합물을 DNeasy Mini 스핀 컬럼으로 이동하여 8,000 rpm에서 1 분간 원심 분리를 실시한 후, 수집 튜브를 새로운 것으로 교체하였다. Buffer AW1 500 μl을 추가한 후, 8000 rpm에서 1 분간 원심 분리를 실시하고, 수집 튜브를 새로운 것으로 교체하였다. 마찬가지로 Buffer AW2 500 μl을 추가한 후, 8000 rpm에서 1 분간 원심 분리를 실시하였으며, 통과물을 버리고 DNeasy Mini 스핀 컬럼을 수집 튜브에 다시 삽입하였다. 최대 속도에서 1 분간 원심 분리한 후, DNeasy Mini 스핀 컬럼을 새로운 1.5 ml 튜브에 놓았다. buffer AE 50 μl을 추가하여 실온에서 1 분간 인큐베이션한 후, 최대 속도에서 1 분간 원심 분리를 진행하였다.The tissues were placed in 1.5 ml microtubes (Axygen), 3 mm tungsten carbide beads, 20 μl of proteinase K, and 200 μl of ATL buffer were added, and the Tissuelyzer was operated at 25 Hz for 3 minutes. Incubation was then performed at 56°C for 20 minutes. Subsequent processes were performed using Qiagen's DNeasy Blood & Tissue mini kits according to the manufacturer's instructions, and the detailed methods are as follows. After adding 20 μl of RNase and 200 μl of AL buffer, the tube was mixed thoroughly. 100% ethanol was added to the sample and mixed thoroughly. The mixture was transferred to a DNeasy Mini spin column, centrifuged at 8,000 rpm for 1 minute, and the collection tube was replaced with a new one. After adding 500 μl of Buffer AW1, centrifugation was performed at 8000 rpm for 1 minute, and the collection tube was replaced with a new one. Similarly, after adding 500 μl of Buffer AW2, centrifugation was performed at 8000 rpm for 1 minute, the flow-through was discarded, and the DNeasy Mini spin column was reinserted into the collection tube. After centrifugation at maximum speed for 1 minute, the DNeasy Mini spin column was placed in a new 1.5 ml tube. After adding 50 μl of Buffer AE, incubation was performed at room temperature for 1 minute, and centrifugation was performed at maximum speed for 1 minute.

실시예 3. 에드워드 증후군 진단을 위한 바이오마커 선정Example 3. Selection of biomarkers for diagnosing Edwards syndrome

에드워드 증후군 진단을 위한 바이오마커 선정을 위해, 혈액 또는 태반 유전체 추출 후 methyl capture sequencing(MC-seq) 분석을 수행하였다.To select biomarkers for diagnosing Edwards syndrome, methyl capture sequencing (MC-seq) analysis was performed after blood or placental genome extraction.

실시예 1 및 2에서 추출한 혈액 및 태반 유전체를 MC-seq을 이용하여 분석하였다. Blood and placental genomes extracted in Examples 1 and 2 were analyzed using MC-seq.

구체적으로, CpG 영역을 갖는 유전자 또는 이의 단편의 CpG 영역의 메틸화 수준을 MC-seq을 이용하여 분석하였다. Specifically, the methylation level of the CpG region of a gene or fragment thereof having a CpG region was analyzed using MC-seq.

에드워드 증후군 진단을 위한 바이오마커 선정을 위해, 혈액 또는 태반 유전체 추출 후 추출된 혈액과 조직의 genomic DNA integrity는 아가로스 젤 전기영동을 실행하여 확인되었으며, Quant-IT PicoGreen (Invitrogen)을 사용하여 양적으로 측정되었다. MC-seq 라이브러리는 SureSelectXT Methyl-Seq Library Preparation kit (Agilent Technologies, 독일)를 이용하여 준비하였다. To select biomarkers for Edwards syndrome diagnosis, genomic DNA integrity of the blood and tissues extracted after blood or placental genome extraction was confirmed by agarose gel electrophoresis and quantitatively measured using Quant-IT PicoGreen (Invitrogen). MC-seq libraries were prepared using the SureSelectXT Methyl-Seq Library Preparation kit (Agilent Technologies, Germany).

이 과정은 다음과 같다. 3mg의 genomic DNA를 Covaris LE220 focused-ultrasonicator (Covaris, Woburn, MA)를 사용하여 150-200 bp가 되도록 파편화 한 후, SureSelect Methyl-Seq Methylated Adapter를 잘려진 단편 DNA에 연결한다. 메틸화된 어댑터가 연결된 DNA는 TapeStation DNA screentape D1000 (Agilent)을 사용하여 양적으로 측정된다. MC-seq을 위해 Agilent SureSelectXT Methyl-Seq Target Enrichment 프로토콜에 따라 350 ng의 DNA 라이브러리가 혼합용 버퍼, 차단 혼합물, RNase block 및 5 μL의 SureSelect Methyl-Seq Capture Library와 혼합된다. Capture baits에 대한 혼성화는 PCR 기계에서 105°C에서 가열된 열 순환기 lid 옵션을 사용하여 65°C에서 24시간 동안 수행된다. 포획된 메틸 DNA는 그 후 세척하여 증폭하고, 최종 정제된 DNAs는 qPCR 정량 프로토콜 가이드(Illumina 시퀀싱 플랫폼용 KAPA 라이브러리 정량 키트)에 따라 qPCR을 사용하여 정량화하고 TapeStation DNA 스크린테이프 HSD1000(Agilent)을 사용하여 정성 검증을 진행한다. 그 후 NovaSeq 플랫폼 (Illumina, San Diego, USA)을 사용하여 시퀀싱한다.Here’s how it works: 3 mg of genomic DNA is fragmented to 150–200 bp using a Covaris LE220 focused-ultrasonicator (Covaris, Woburn, MA), and SureSelect Methyl-Seq Methylated Adapters are ligated to the sheared DNA fragments. The methylated adapter-ligated DNA is quantified using TapeStation DNA screentape D1000 (Agilent). For MC-seq, 350 ng of DNA library is mixed with mixing buffer, blocking mixture, RNase block, and 5 μL of SureSelect Methyl-Seq Capture Library according to the Agilent SureSelectXT Methyl-Seq Target Enrichment protocol. Hybridization to the Capture baits is performed at 65°C for 24 h using a thermocycler lid option heated at 105°C in a PCR machine. The captured methyl DNA is then washed and amplified, and the final purified DNAs are quantified using qPCR according to the qPCR quantification protocol guide (KAPA Library Quantification Kit for Illumina Sequencing Platforms) and qualitatively verified using TapeStation DNA Screen Tape HSD1000 (Agilent). They are then sequenced using the NovaSeq platform (Illumina, San Diego, USA).

이후 생성된 데이터 처리 및 메틸레이션 calling 방법은 아래와 같이 수행한다. 시퀀싱에서 생성된 paired-end read (101 bp)는 FastQC (버전 0.11.7)를 사용하여 시퀀스 품질을 확인하고, 분석 시작 전에 Trimmomatic (버전 0.38)을 사용하여 어댑터 시퀀스와 품질이 3 미만인 베이스를 read 끝에서 제거한다. 또한 슬라이딩 윈도우 트리밍 방법을 사용하여 창 크기 = 4 및 평균 품질 = 15에 적합하지 않은 베이스를 제거하고 최소 길이 36bp의 read를 제거하여 정제된 데이터를 생성한다. 정제된 read는 BSMAP을 사용하여 인간 게놈 (UCSC hg19)에 맞추고 BSMAP (버전 2.90 매개변수 설정 -n 1 -r 0)은 유일하게 매핑된 read만 허용하여 매핑된 데이터 (SAM 파일 형식)는 sambamba (버전 0.6.5)를 통해 정렬 및 인덱싱한다. 그 후에 sambamba (버전 0.6.5)를 사용하여 PCR 중복을 제거한다. 메틸레이션 수준은 BSMAP 프로그램의 methratio.py를 사용하여 calling하고, Agilent SureSelect 대상 영역에 위치한 모든 단일 시토신의 메틸레이션 calling을 수행하며, 결과 커버리지 프로필은 각각의 세 시퀀스 컨텍스트 (CG, CHG 및 CHH)에 대해 # of C / 유효한 CT 카운트로 해석한다.Hereinafter, the generated data processing and methylation calling method are performed as follows. The paired-end reads (101 bp) generated from sequencing are checked for sequence quality using FastQC (version 0.11.7), and adapter sequences and bases with quality less than 3 are removed from the end of the reads using Trimmomatic (version 0.38) before starting the analysis. In addition, the sliding window trimming method is used to remove bases that do not fit the window size = 4 and average quality = 15, and reads with a minimum length of 36 bp are removed to generate refined data. The refined reads are aligned to the human genome (UCSC hg19) using BSMAP, and BSMAP (version 2.90 parameter setting -n 1 -r 0) allows only uniquely mapped reads, and the mapped data (SAM file format) is aligned and indexed via sambamba (version 0.6.5). After that, PCR duplicates are removed using sambamba (version 0.6.5). Methylation levels are called using methratio.py from the BSMAP program, which performs methylation calling of all single cytosines located in the Agilent SureSelect target region, and the resulting coverage profiles are interpreted as # of C/valid CT counts for each of the three sequence contexts (CG, CHG, and CHH).

분석에 있어서, CpG site의 메틸화 수준은 0 내지 100으로 표현되며 0은 비메틸화, 100은 완전한 메틸화를 의미한다.In analysis, the methylation level of a CpG site is expressed as 0 to 100, with 0 indicating unmethylation and 100 indicating complete methylation.

바이오마커 선정에 있어서, 에드워드 증후군 태아의 태반에서 나타나는 후성학적 특징에 따라 과메틸화 마커와 저메틸화된 마커로 구분하여 바이오마커를 발굴하였다. 동일한 형태의 후성학적 특성이 마커 부위를 포함하여 연속적으로 2개 이상 존재하는 부위를 선정하였다. 구체적으로, 모체, 정상 태아 및 에드워드 증후군 태아 순으로 CpG 영역의 메틸화 수준이 증가하거나 또는 감소하는 후성학적 특성이 연속적으로 2개 이상 존재하는 부위로서, 상기 후성학적 특성이 유지되는 부위를 가지는 유전자 또는 이의 단편을 바이오마커로 선정하였다.In selecting biomarkers, biomarkers were discovered by dividing them into hypermethylated markers and hypomethylated markers according to the epigenetic characteristics shown in the placenta of Edwards syndrome fetuses. Regions where two or more epigenetic characteristics of the same type exist consecutively, including the marker region, were selected. Specifically, a gene or a fragment thereof having a region where the methylation level of the CpG region increases or decreases in the order of the mother, normal fetus, and Edwards syndrome fetus, and where the epigenetic characteristics are maintained, were selected as biomarkers.

3.1. 에드워드 증후군 진단을 위한 과메틸화 바이오마커 선정3.1. Selection of hypermethylation biomarkers for the diagnosis of Edwards syndrome

모체 혈액보다 정상 태아 태반에서 메틸화 수준이 높고, 정상 태아 태반보다 에드워드 증후군 태아 태반에서 메틸화 수준이 높은 유전자 또는 이의 단편을 과메틸화 바이오마커로 선정하였다. 구체적으로, 선정된 과메틸화 바이오마커는 모체 혈액에서는 0.2 이하의 낮은 메틸화 수준을 나타내며, 정상 태아에서는 모체 혈액에 비해 △ + 0.2 ~ + 0.3 수준의 높은 과메틸화를 나타내고, 에드워드 증후군 태아에서는 정상 태아에 비해 △ + 0.2 ~ + 0.3 수준의 높은 과메틸화를 나타낸다.A gene or a fragment thereof that has a higher methylation level in the normal fetal placenta than in the maternal blood, and a higher methylation level in the Edwards syndrome fetal placenta than in the normal fetal placenta, was selected as a hypermethylation biomarker. Specifically, the selected hypermethylation biomarker shows a low methylation level of 0.2 or less in the maternal blood, a high hypermethylation of △ + 0.2 to + 0.3 in the normal fetus compared to the maternal blood, and a high hypermethylation of △ + 0.2 to + 0.3 in the Edwards syndrome fetus compared to the normal fetus.

선정된 67개의 과메틸화 바이오마커를 하기 표 3에 나타내었다.The 67 selected hypermethylation biomarkers are shown in Table 3 below.

유전자gene bpbp CG 개수CG count 유전자 내 영역Region within a gene MXRA8MXRA8 685685 6464 chr1-1289873-1290557chr1-1289873-1290557 PTPRUPTPRU 237237 1919 chr1-29585903-29586139chr1-29585903-29586139 IL12RB2IL12RB2 250250 99 chr1-67773330-67773579chr1-67773330-67773579 WNT3AWNT3A 387387 1717 chr1-228225569-228225955chr1-228225569-228225955 KIF26BKIF26B 100100 1010 chr1-245851386-245851485chr1-245851386-245851485 SLC30A3/DNAJC5GSLC30A3/DNAJC5G 110110 99 chr2-27487380-27487489chr2-27487380-27487489 ZFP36L2ZFP36L2 113113 1010 chr2-43451900-43452012chr2-43451900-43452012 ZIC4ZIC4 115115 77 chr3-147113790-147113904chr3-147113790-147113904 CPLX1CPLX1 156156 44 chr4-789186-789341chr4-789186-789341 ZFYVE28ZFYVE28 209209 66 chr4-2366608-2366816chr4-2366608-2366816 CCNG2/CXCL13CCNG2/CXCL13 231231 1010 chr4-78114672-78114902chr4-78114672-78114902 PALLDPALLD 161161 44 chr4-169799215-169799375chr4-169799215-169799375 IRX4IRX4 137137 1010 chr5-1878570-1878706chr5-1878570-1878706 C5orf38C5orf38 173173 2727 chr5-2755271-2755443chr5-2755271-2755443 ISL1ISL1 331331 1919 chr5-50685660-50685990chr5-50685660-50685990 LHFPL2LHFPL2 226226 1616 chr5-77805683-77805908chr5-77805683-77805908 GPRIN1GPRIN1 462462 2525 chr5-176023779-176024240chr5-176023779-176024240 LOC100506207/TFAP2ALOC100506207/TFAP2A 149149 1212 chr6-10385242-10385390chr6-10385242-10385390 GFOD1/SIRT5GFOD1/SIRT5 280280 44 chr6-13561982-13562261chr6-13561982-13562261 ETV7ETV7 305305 1414 chr6-36355849-36356153chr6-36355849-36356153 FOXP4FOXP4 153153 1111 chr6-41528771-41528923chr6-41528771-41528923 SMOC2SMOC2 702702 3737 chr6-168841499-168842200chr6-168841499-168842200 GNA12/CARD11GNA12/CARD11 153153 66 chr7-2903209-2903361chr7-2903209-2903361 AP5Z1AP5Z1 166166 1111 chr7-4832070-4832235chr7-4832070-4832235 ZNF273/ZNF117ZNF273/ZNF117 187187 1919 chr7-64408037-64408223chr7-64408037-64408223 CALN1CALN1 161161 1414 chr7-71801254-71801414chr7-71801254-71801414 SND1SND1 116116 88 chr7-127671835-127671950chr7-127671835-127671950 HTR5A/INSIG1HTR5A/INSIG1 817817 1010 chr7-154997216-154998032chr7-154997216-154998032 MNX1MNX1 237237 77 chr7-156798050-156798286chr7-156798050-156798286 EBF2/PPP2R2AEBF2/PPP2R2A 156156 77 chr8-25907653-25907808chr8-25907653-25907808 FAM110BFAM110B 316316 1616 chr8-59058025-59058340chr8-59058025-59058340 OSR2/VPS13BOSR2/VPS13B 108108 88 chr8-99986018-99986125chr8-99986018-99986125 NAPRTNAPRT 889889 8989 chr8-144659885-144660773chr8-144659885-144660773 GLIS3GLIS3 158158 33 chr9-4299929-4300086chr9-4299929-4300086 SLC24A2/MLLT3SLC24A2/MLLT3 109109 1010 chr9-19934446-19934554chr9-19934446-19934554 CRB2CRB2 145145 1313 chr9-126135490-126135634chr9-126135490-126135634 RABL6RABL6 130130 1010 chr9-139715867-139715996chr9-139715867-139715996 TACC2TACC2 337337 1717 chr10-123923101-123923437chr10-123923101-123923437 CPXM2/CHST15CPXM2/CHST15 127127 77 chr10-125751219-125751345chr10-125751219-125751345 CLMPCLMP 148148 99 chr11-123066597-123066744chr11-123066597-123066744 KRT7/KRT81KRT7/KRT81 185185 2121 chr12-52652331-52652515chr12-52652331-52652515 HOXC4HOXC4 284284 77 chr12-54447318-54447601chr12-54447318-54447601 LOC100287944LOC100287944 101101 55 chr12-106988280-106988380chr12-106988280-106988380 LINC00403LINC00403 109109 99 chr13-112707765-112707873chr13-112707765-112707873 LINC00403LINC00403 228228 1616 chr13-112711235-112711462chr13-112711235-112711462 LINC00925LINC00925 140140 99 chr15-89921078-89921217chr15-89921078-89921217 NR2F2-AS1NR2F2-AS1 278278 66 chr15-96866980-96867257chr15-96866980-96867257 MGRN1MGRN1 106106 1212 chr16-4714056-4714161chr16-4714056-4714161 RBFOX1RBFOX1 102102 1111 chr16-6069329-6069430chr16-6069329-6069430 PLEKHH3PLEKHH3 248248 1010 chr17-40822053-40822300chr17-40822053-40822300 EMILIN2EMILIN2 156156 66 chr18-2905913-2906068chr18-2905913-2906068 ST8SIA3ST8SIA3 278278 1212 chr18-55020377-55020654chr18-55020377-55020654 ST8SIA3/ONECUT2ST8SIA3/ONECUT2 134134 88 chr18-55094946-55095079chr18-55094946-55095079 ST8SIA3/ONECUT2ST8SIA3/ONECUT2 151151 1010 chr18-55096004-55096154chr18-55096004-55096154 ST8SIA3/ONECUT2ST8SIA3/ONECUT2 119119 44 chr18-55097474-55097592chr18-55097474-55097592 ONECUT2ONECUT2 160160 1515 chr18-55101950-55102109chr18-55101950-55102109 NFATC1/CTDP1NFATC1/CTDP1 123123 2222 chr18-77397555-77397677chr18-77397555-77397677 PARD6GPARD6G 163163 1414 chr18-77917687-77917849chr18-77917687-77917849 ZNF563ZNF563 272272 1616 chr19-12444251-12444522chr19-12444251-12444522 MED29/ZFP36MED29/ZFP36 185185 88 chr19-39894639-39894823chr19-39894639-39894823 DYRK1BDYRK1B 716716 3232 chr19-40324031-40324746chr19-40324031-40324746 PPP1R13LPPP1R13L 391391 1212 chr19-45888913-45889303chr19-45888913-45889303 CYP24A1CYP24A1 185185 1717 chr20-52789641-52789825chr20-52789641-52789825 CYP24A1CYP24A1 181181 77 chr20-52790137-52790317chr20-52790137-52790317 C21orf2/TRPM2C21orf2/TRPM2 151151 88 chr21-45770053-45770203chr21-45770053-45770203 YBEYYBEY 111111 66 chr21-47717820-47717930chr21-47717820-47717930 MAPK8IP2MAPK8IP2 273273 1010 chr22-51042493-51042765chr22-51042493-51042765

정상 모체 혈액, 정상 태아 태반 및 에드워드 증후군 태아 태반에서 상기 선정된 유전자 영역의 메틸화 정도 (메틸화수준 (%)을 100으로 나눈 값)를 하기 도 1, 도 2 및 표 4에 나타내었다.The methylation levels (methylation level (%) divided by 100) of the selected gene regions in normal maternal blood, normal fetal placenta, and Edwards syndrome fetal placenta are shown in Figures 1, 2, and Table 4 below.

도 1 및 도 2는 에드워드 증후군 진단용으로 선정된 에드워드 증후군 특이적 과메틸화 바이오마커의 수준을 히트맵(heat map)분석한 결과이다.Figures 1 and 2 show the results of a heat map analysis of the levels of Edwards syndrome-specific hypermethylation biomarkers selected for the diagnosis of Edwards syndrome.

상기 도면에서 NB는 모체 혈액, NT는 정상 태아의 태반, T18T는 에드워드 증후군 태아의 태반을 나타낸다.In the above figure, NB represents maternal blood, NT represents the placenta of a normal fetus, and T18T represents the placenta of an Edwards syndrome fetus.

유전자gene 메틸화 정도Degree of methylation 정상 모체 혈액normal maternal blood 정상 태아 태반Normal fetal placenta 에드워드증후군 태아 태반Edwards syndrome fetal placenta MXRA8MXRA8 0.150.15 0.480.48 0.670.67 PTPRUPTPRU 0.020.02 0.270.27 0.490.49 IL12RB2IL12RB2 0.020.02 0.280.28 0.520.52 WNT3AWNT3A 0.020.02 0.090.09 0.390.39 KIF26BKIF26B 0.130.13 0.370.37 0.560.56 SLC30A3/DNAJC5GSLC30A3/DNAJC5G 0.020.02 0.200.20 0.440.44 ZFP36L2ZFP36L2 0.030.03 0.330.33 0.560.56 ZIC4ZIC4 0.020.02 0.340.34 0.610.61 CPLX1CPLX1 0.040.04 0.300.30 0.560.56 ZFYVE28ZFYVE28 0.160.16 0.470.47 0.640.64 CCNG2/CXCL13CCNG2/CXCL13 0.020.02 0.390.39 0.570.57 PALLDPALLD 0.010.01 0.330.33 0.560.56 IRX4IRX4 0.100.10 0.490.49 0.720.72 C5orf38C5orf38 0.040.04 0.290.29 0.480.48 ISL1ISL1 0.040.04 0.360.36 0.520.52 LHFPL2LHFPL2 0.020.02 0.280.28 0.450.45 GPRIN1GPRIN1 0.020.02 0.200.20 0.430.43 LOC100506207/TFAP2ALOC100506207/TFAP2A 0.120.12 0.270.27 0.440.44 GFOD1/SIRT5GFOD1/SIRT5 0.180.18 0.430.43 0.650.65 ETV7ETV7 0.090.09 0.290.29 0.490.49 FOXP4FOXP4 0.010.01 0.330.33 0.520.52 SMOC2SMOC2 0.040.04 0.310.31 0.570.57 GNA12/CARD11GNA12/CARD11 0.020.02 0.420.42 0.580.58 AP5Z1AP5Z1 0.100.10 0.560.56 0.780.78 ZNF273/ZNF117ZNF273/ZNF117 0.030.03 0.520.52 0.710.71 CALN1CALN1 0.010.01 0.260.26 0.480.48 SND1SND1 0.010.01 0.260.26 0.550.55 HTR5A/INSIG1HTR5A/INSIG1 0.030.03 0.340.34 0.520.52 MNX1MNX1 0.010.01 0.310.31 0.510.51 EBF2/PPP2R2AEBF2/PPP2R2A 0.020.02 0.380.38 0.570.57 FAM110BFAM110B 0.020.02 0.340.34 0.560.56 OSR2/VPS13BOSR2/VPS13B 0.080.08 0.560.56 0.720.72 NAPRTNAPRT 0.040.04 0.180.18 0.550.55 GLIS3GLIS3 0.010.01 0.350.35 0.580.58 SLC24A2/MLLT3SLC24A2/MLLT3 0.090.09 0.360.36 0.530.53 CRB2CRB2 0.090.09 0.440.44 0.620.62 RABL6RABL6 0.240.24 0.500.50 0.730.73 TACC2TACC2 0.020.02 0.300.30 0.560.56 CPXM2/CHST15CPXM2/CHST15 0.060.06 0.380.38 0.640.64 CLMPCLMP 0.060.06 0.340.34 0.550.55 KRT7/KRT81KRT7/KRT81 0.020.02 0.360.36 0.560.56 HOXC4HOXC4 0.030.03 0.430.43 0.600.60 LOC100287944LOC100287944 0.070.07 0.280.28 0.500.50 LINC00403LINC00403 0.080.08 0.400.40 0.560.56 LINC00403LINC00403 0.060.06 0.360.36 0.550.55 LINC00925LINC00925 0.140.14 0.430.43 0.560.56 NR2F2-AS1NR2F2-AS1 0.240.24 0.550.55 0.680.68 MGRN1MGRN1 0.170.17 0.580.58 0.700.70 RBFOX1RBFOX1 0.060.06 0.290.29 0.460.46 PLEKHH3PLEKHH3 0.120.12 0.610.61 0.870.87 EMILIN2EMILIN2 0.060.06 0.300.30 0.500.50 ST8SIA3ST8SIA3 0.060.06 0.300.30 0.490.49 ST8SIA3/ONECUT2ST8SIA3/ONECUT2 0.050.05 0.330.33 0.560.56 ST8SIA3/ONECUT2ST8SIA3/ONECUT2 0.080.08 0.490.49 0.640.64 ST8SIA3/ONECUT2ST8SIA3/ONECUT2 0.080.08 0.500.50 0.670.67 ONECUT2ONECUT2 0.030.03 0.320.32 0.490.49 NFATC1/CTDP1NFATC1/CTDP1 0.100.10 0.470.47 0.590.59 PARD6GPARD6G 0.290.29 0.560.56 0.780.78 ZNF563ZNF563 0.010.01 0.510.51 0.730.73 MED29/ZFP36MED29/ZFP36 0.030.03 0.350.35 0.520.52 DYRK1BDYRK1B 0.010.01 0.340.34 0.530.53 PPP1R13LPPP1R13L 0.090.09 0.420.42 0.610.61 CYP24A1CYP24A1 0.020.02 0.330.33 0.490.49 CYP24A1CYP24A1 0.020.02 0.340.34 0.480.48 C21orf2/TRPM2C21orf2/TRPM2 0.030.03 0.240.24 0.460.46 YBEYYBEY 0.090.09 0.480.48 0.630.63 MAPK8IP2MAPK8IP2 0.030.03 0.280.28 0.490.49

도 1, 도 2 및 표 4에 나타낸 바와 같이, MXRA8, PTPRU, IL12RB2, WNT3A, KIF26B,SLC30A3/DNAJC5G, ZFP36L2, ZIC4, CPLX1, ZFYVE28, CCNG2/CXCL13, PALLD, IRX4, C5orf38, ISL1, LHFPL2, GPRIN1, LOC100506207/TFAP2A, GFOD1/SIRT5, ETV7, FOXP4, SMOC2, GNA12/CARD11, AP5Z1, ZNF273/ZNF117, CALN1, SND1, HTR5A/INSIG1, MNX1, EBF2/PPP2R2A, FAM110B, OSR2/VPS13B, NAPRT, GLIS3, SLC24A2/MLLT3, CRB2, RABL6, TACC2, CPXM2/CHST15, CLMP, KRT7/KRT81, HOXC4, LOC100287944, LINC00403, LINC00925, NR2F2-AS1, MGRN1, RBFOX1, PLEKHH3, EMILIN2, ST8SIA3, ST8SIA3/ONECUT2, ONECUT2, NFATC1/CTDP1, PARD6G, ZNF563, MED29/ZFP36, DYRK1B, PPP1R13L, CYP24A1, C21orf2/TRPM2, YBEY 및 MAPK8IP2 의 CpG 영역을 갖는 단편의 경우, 모체 혈액보다 정상 태아 태반에서 CpG 영역의 높은 메틸화 수준을 나타냈고, 정상 태아 태반보다 에드워드 증후군 태아 태반에서 CpG 영역의 높은 메틸화 수준을 나타냈다. As shown in FIG. 1, FIG. 2 and Table 4, MXRA8, PTPRU, IL12RB2, WNT3A, KIF26B, SLC30A3/DNAJC5G, ZFP36L2, ZIC4, CPLX1, ZFYVE28, CCNG2/CXCL13, PALLD, IRX4, C5orf38, ISL1, LHFPL2, GPRIN1, LOC100506207/TFAP2A, GFOD1/SIRT5, ETV7, FOXP4, SMOC2, GNA12/CARD11, AP5Z1, ZNF273/ZNF117, CALN1, SND1, HTR5A/INSIG1, MNX1, EBF2/PPP2R2A, FAM110B, OSR2/VPS13B, NAPRT, GLIS3, For fragments with CpG regions of SLC24A2/MLLT3, CRB2, RABL6, TACC2, CPXM2/CHST15, CLMP, KRT7/KRT81, HOXC4, LOC100287944, LINC00403, LINC00925, NR2F2-AS1, MGRN1, RBFOX1, PLEKHH3, EMILIN2, ST8SIA3, ST8SIA3/ONECUT2, ONECUT2, NFATC1/CTDP1, PARD6G, ZNF563, MED29/ZFP36, DYRK1B, PPP1R13L, CYP24A1, C21orf2/TRPM2, YBEY , and MAPK8IP2 , higher methylation levels of CpG regions were observed in normal fetal placenta than in maternal blood, and higher methylation levels were observed in Edwards blood than in normal fetal placenta. High methylation levels of CpG regions were observed in the placenta of the fetuses with the syndrome.

상기 67개의 유전자(또는 이의 단편)을 과메틸화 바이오마커로 선정하였으며, 상기 바이오마커에 있어서, CpG site의 메틸화 수준이 정상 태아 태반이 모체 혈액보다 10 내지 50 정도 높았으며, 에드워드 증후군 태아 태반이 정상 태아 태반 보다 10 내지 50 정도 높았다.The above 67 genes (or fragments thereof) were selected as hypermethylation biomarkers, and the methylation level of the CpG site in the above biomarkers was about 10 to 50 higher in the normal fetal placenta than in the maternal blood, and about 10 to 50 higher in the Edwards syndrome fetal placenta than in the normal fetal placenta.

상기 바이오마커에 있어서, 에드워드 증후군 태아 태반과 다른 두 그룹 간의 메틸화 수준의 차이는 모두 통계적으로 유의하였다(p value < 0.05).For the above biomarkers, the differences in methylation levels between the Edwards syndrome fetal placentas and the other two groups were all statistically significant (p value < 0.05).

3.2. 에드워드 증후군 진단을 위한 저메틸화 바이오마커 선정3.2. Selection of hypomethylated biomarkers for the diagnosis of Edwards syndrome

모체 혈액보다 정상 태아 태반에서 메틸화 수준이 낮고, 정상 태아 태반보다 에드워드 증후군 태아 태반에서 메틸화 수준이 낮은 유전자 또는 이의 단편을 저메틸화 바이오마커로 선정하였다. 구체적으로, 선정된 저메틸화 바이오마커는 모체 혈액에서는 0.8 이상의 높은 메틸화 수준을 나타내며, 정상 태아에서는 모체 혈액에 비해 △ - 0.4 ~ - 0.5 수준의 낮은 저메틸화를 나타내고, 에드워드 증후군 태아에서는 정상 태아에 비해 △- 0.2 ~ - 0.4 수준의 낮은 저메틸화를 나타낸다.A gene or a fragment thereof that has a lower methylation level in the normal fetal placenta than in the maternal blood and a lower methylation level in the Edwards syndrome fetal placenta than in the normal fetal placenta was selected as a hypomethylation biomarker. Specifically, the selected hypomethylation biomarker shows a high methylation level of 0.8 or higher in the maternal blood, a low hypomethylation of △ - 0.4 to - 0.5 in the normal fetus compared to the maternal blood, and a low hypomethylation of △ - 0.2 to - 0.4 in the Edwards syndrome fetus compared to the normal fetus.

선정된 91개의 저메틸화 바이오마커를 하기 표 5에 나타내었다.The 91 selected hypomethylated biomarkers are shown in Table 5 below.

유전자gene bpbp CG 개수CG count 유전자 내 영역Region within a gene LCKLCK 136136 44 chr1-32742005-32742140chr1-32742005-32742140 FCRL5FCRL5 151151 22 chr1-157492943-157493093chr1-157492943-157493093 RHOU/RAB4ARHOU/RAB4A 277277 33 chr1-229374459-229374735chr1-229374459-229374735 GREM2/RGS7GREM2/RGS7 180180 44 chr1-240817303-240817482chr1-240817303-240817482 DCDC2C/LINC01304DCDC2C/LINC01304 106106 44 chr2-3990335-3990440chr2-3990335-3990440 RNU6-81PRNU6-81P 142142 33 chr2-132300645-132300786chr2-132300645-132300786 CCDC108CCDC108 151151 11 chr2-219892467-219892617chr2-219892467-219892617 LOC150935/MIR4786LOC150935/MIR4786 201201 11 chr2-240752453-240752653chr2-240752453-240752653 AQP12BAQP12B 178178 99 chr2-241622219-241622396chr2-241622219-241622396 AQP12AAQP12A 151151 11 chr2-241631133-241631283chr2-241631133-241631283 LINC01237LINC01237 151151 44 chr2-242841595-242841745chr2-242841595-242841745 XCR1XCR1 201201 11 chr3-46069296-46069496chr3-46069296-46069496 FGFR3FGFR3 117117 44 chr4-1805102-1805218chr4-1805102-1805218 NDST4/MIR1973NDST4/MIR1973 201201 22 chr4-117218386-117218586chr4-117218386-117218586 TPPPTPPP 178178 33 chr5-669788-669965chr5-669788-669965 HNRNPH1/C5orf60HNRNPH1/C5orf60 274274 1212 chr5-179059331-179059604chr5-179059331-179059604 RASGEF1CRASGEF1C 158158 22 chr5-179528232-179528389chr5-179528232-179528389 LINC01012/LOC100131289LINC01012/LOC100131289 205205 55 chr6-27706844-27707048chr6-27706844-27707048 HLA-DPA1HLA-DPA1 143143 22 chr6-33032739-33032881chr6-33032739-33032881 HLA-DPA1HLA-DPA1 146146 33 chr6-33035235-33035380chr6-33035235-33035380 HLA-DPA1HLA-DPA1 101101 11 chr6-33036446-33036546chr6-33036446-33036546 HLA-DPA1HLA-DPA1 165165 33 chr6-33037577-33037741chr6-33037577-33037741 SMOC2/THBS2SMOC2/THBS2 262262 66 chr6-169539688-169539949chr6-169539688-169539949 ADAP1ADAP1 125125 44 chr7-987360-987484chr7-987360-987484 ELFN1ELFN1 101101 66 chr7-1786348-1786448chr7-1786348-1786448 MAD1L1MAD1L1 179179 33 chr7-1891369-1891547chr7-1891369-1891547 LYPD2LYPD2 555555 55 chr8-143834030-143834584chr8-143834030-143834584 MAPK15MAPK15 165165 33 chr8-144801594-144801758chr8-144801594-144801758 PUF60PUF60 163163 22 chr8-144906207-144906369chr8-144906207-144906369 LINGO2/LINC01242LINGO2/LINC01242 151151 44 chr9-29826505-29826655chr9-29826505-29826655 APBA1APBA1 156156 44 chr9-72131795-72131950chr9-72131795-72131950 NCS1NCS1 145145 55 chr9-132955955-132956099chr9-132955955-132956099 FIBCD1FIBCD1 202202 44 chr9-133779363-133779564chr9-133779363-133779564 FIBCD1FIBCD1 214214 66 chr9-133779750-133779963chr9-133779750-133779963 FIBCD1FIBCD1 174174 88 chr9-133799133-133799306chr9-133799133-133799306 RXRA/COL5A1RXRA/COL5A1 166166 33 chr9-137356284-137356449chr9-137356284-137356449 KCNT1KCNT1 125125 22 chr9-138669335-138669459chr9-138669335-138669459 GPSM1GPSM1 209209 44 chr9-139230380-139230588chr9-139230380-139230588 PFKFB3PFKFB3 101101 33 chr10-6264447-6264547chr10-6264447-6264547 ATRNL1ATRNL1 463463 22 chr10-117668266-117668728chr10-117668266-117668728 FRG2B/DUX4L3FRG2B/DUX4L3 193193 44 chr10-135453991-135454183chr10-135453991-135454183 PKP3PKP3 178178 66 chr11-394531-394708chr11-394531-394708 MUC5BMUC5B 151151 55 chr11-1263116-1263266chr11-1263116-1263266 LMO1LMO1 155155 33 chr11-8248450-8248604chr11-8248450-8248604 LOC441601LOC441601 599599 5454 chr11-50257274-50257872chr11-50257274-50257872 APOA5APOA5 282282 1010 chr11-116661162-116661443chr11-116661162-116661443 TRIM29TRIM29 321321 44 chr11-119992252-119992572chr11-119992252-119992572 B4GALNT3B4GALNT3 180180 77 chr12-670471-670650chr12-670471-670650 CACNA2D4LRTM2CACNA2D4LRTM2 295295 55 chr12-1936192-1936486chr12-1936192-1936486 TSPAN9/PRMT8TSPAN9/PRMT8 205205 44 chr12-3476351-3476555chr12-3476351-3476555 VAMP1/MRPL51VAMP1/MRPL51 149149 33 chr12-6586920-6587068chr12-6586920-6587068 GNB3GNB3 250250 33 chr12-6949911-6950160chr12-6949911-6950160 LRIG3/SLC16A7LRIG3/SLC16A7 427427 44 chr12-59924804-59925230chr12-59924804-59925230 CABP1CABP1 183183 44 chr12-121104906-121105088chr12-121104906-121105088 TMEM132DTMEM132D 166166 33 chr12-130334337-130334502chr12-130334337-130334502 LINC01257LINC01257 145145 33 chr12-131648973-131649117chr12-131648973-131649117 LOC101928416/FBRSL1LOC101928416/FBRSL1 189189 44 chr12-133009259-133009447chr12-133009259-133009447 LINC00572LINC00572 123123 33 chr13-30498612-30498734chr13-30498612-30498734 MCF2LMCF2L 132132 22 chr13-113732740-113732871chr13-113732740-113732871 MCF2LMCF2L 181181 55 chr13-113732999-113733179chr13-113732999-113733179 GRTP1/ADPRHL1GRTP1/ADPRHL1 331331 77 chr13-114064805-114065135chr13-114064805-114065135 EAPP/SNX6EAPP/SNX6 197197 88 chr14-35021946-35022142chr14-35021946-35022142 ELK2AP/KIAA0125ELK2AP/KIAA0125 151151 77 chr14-106145858-106146008chr14-106145858-106146008 ADAM6/LINC00226ADAM6/LINC00226 151151 22 chr14-106539847-106539997chr14-106539847-106539997 LINC00226/LINC00221LINC00226/LINC00221 154154 44 chr14-106857323-106857476chr14-106857323-106857476 C15orf48C15orf48 173173 44 chr15-45723131-45723303chr15-45723131-45723303 SV2BSV2B 148148 1212 chr15-91840105-91840252chr15-91840105-91840252 LOC101927332/FAM169BLOC101927332/FAM169B 136136 33 chr15-98647977-98648112chr15-98647977-98648112 SBK1SBK1 160160 66 chr16-28332430-28332589chr16-28332430-28332589 RRN3P2/SNX29P2RRN3P2/SNX29P2 153153 55 chr16-29150987-29151139chr16-29150987-29151139 ARHGEF15ARHGEF15 220220 55 chr17-8219200-8219419chr17-8219200-8219419 NFE2L1NFE2L1 190190 33 chr17-46131641-46131830chr17-46131641-46131830 SEC14L1/SEPT9SEC14L1/SEPT9 174174 22 chr17-75276276-75276449chr17-75276276-75276449 B3GNTL1/METRNLB3GNTL1/METRNL 230230 33 chr17-81025581-81025810chr17-81025581-81025810 AQP4-AS1AQP4-AS1 191191 44 chr18-24446445-24446635chr18-24446445-24446635 TSHZ1TSHZ1 257257 44 chr18-72924982-72925238chr18-72924982-72925238 GALR1/LINC01029GALR1/LINC01029 201201 22 chr18-75402175-75402375chr18-75402175-75402375 LINC01029, SALL3LINC01029, SALL3 187187 44 chr18-76690593-76690779chr18-76690593-76690779 GRIN3BGRIN3B 122122 33 chr19-1003445-1003566chr19-1003445-1003566 SMIM24/DOHHSMIM24/DOHH 175175 44 chr19-3482825-3482999chr19-3482825-3482999 C19orf67C19orf67 178178 1010 chr19-14196529-14196706chr19-14196529-14196706 TMC4TMC4 178178 22 chr19-54668914-54669091chr19-54668914-54669091 LILRB3LILRB3 270270 44 chr19-54724045-54724314chr19-54724045-54724314 SIRPG/LOC100289473SIRPG/LOC100289473 129129 55 chr20-1716350-1716478chr20-1716350-1716478 FOXS1FOXS1 208208 22 chr20-30432958-30433165chr20-30432958-30433165 HELZ2HELZ2 106106 55 chr20-62194775-62194880chr20-62194775-62194880 LOC101927797/LINC00320LOC101927797/LINC00320 245245 44 chr21-21272245-21272489chr21-21272245-21272489 LINC00111LINC00111 144144 55 chr21-43109581-43109724chr21-43109581-43109724 TFF2TFF2 171171 33 chr21-43771286-43771456chr21-43771286-43771456 ZDHHC8P1ZDHHC8P1 232232 44 chr22-23732095-23732326chr22-23732095-23732326 LOC284930LOC284930 161161 66 chr22-48027673-48027833chr22-48027673-48027833

정상 모체 혈액, 정상 태아 태반 및 에드워드 증후군 태아 태반에서 상기 선정된 유전자 영역의 메틸화 정도 (메틸화수준 (%)을 100으로 나눈 값)를 하기 도 3 및 도 4 및 표 6에 나타내었다.The methylation levels (methylation level (%) divided by 100) of the selected gene regions in normal maternal blood, normal fetal placenta, and Edwards syndrome fetal placenta are shown in Figures 3 and 4 and Table 6 below.

도 3 및 도 4는 에드워드 증후군 진단용으로 선정된 에드워드 증후군 특이적 저메틸화 바이오마커의 수준을 히트맵(heat map)분석한 결과이다.Figures 3 and 4 show the results of a heat map analysis of the levels of Edwards syndrome-specific hypomethylated biomarkers selected for the diagnosis of Edwards syndrome.

상기 도면에서 NB는 모체 혈액, NT는 정상 태아의 태반, T18T는 에드워드 증후군 태아의 태반을 나타낸다.In the above figure, NB represents maternal blood, NT represents the placenta of a normal fetus, and T18T represents the placenta of an Edwards syndrome fetus.

유전자gene 메틸화 정도Degree of methylation 정상 모체 혈액normal maternal blood 정상 태아 태반Normal fetal placenta 에드워드증후군 태아 태반Edwards syndrome fetal placenta LCKLCK 0.91 0.91 0.53 0.53 0.22 0.22 FCRL5FCRL5 0.79 0.79 0.58 0.58 0.28 0.28 RHOU/RAB4ARHOU/RAB4A 0.85 0.85 0.45 0.45 0.18 0.18 GREM2/RGS7GREM2/RGS7 0.92 0.92 0.69 0.69 0.35 0.35 DCDC2C/LINC01304DCDC2C/LINC01304 0.79 0.79 0.62 0.62 0.41 0.41 RNU6-81PRNU6-81P 0.97 0.97 0.57 0.57 0.30 0.30 CCDC108CCDC108 0.90 0.90 0.53 0.53 0.30 0.30 LOC150935/MIR4786LOC150935/MIR4786 0.81 0.81 0.45 0.45 0.24 0.24 AQP12BAQP12B 0.85 0.85 0.44 0.44 0.25 0.25 AQP12AAQP12A 0.89 0.89 0.46 0.46 0.24 0.24 LINC01237LINC01237 0.90 0.90 0.54 0.54 0.35 0.35 XCR1XCR1 0.86 0.86 0.54 0.54 0.33 0.33 FGFR3FGFR3 0.94 0.94 0.62 0.62 0.41 0.41 NDST4/MIR1973NDST4/MIR1973 0.92 0.92 0.48 0.48 0.26 0.26 TPPPTPPP 0.88 0.88 0.57 0.57 0.38 0.38 HNRNPH1/C5orf60HNRNPH1/C5orf60 0.90 0.90 0.52 0.52 0.33 0.33 RASGEF1CRASGEF1C 0.91 0.91 0.62 0.62 0.35 0.35 LINC01012/LOC100131289LINC01012/LOC100131289 0.92 0.92 0.65 0.65 0.44 0.44 HLA-DPA1HLA-DPA1 0.68 0.68 0.28 0.28 0.02 0.02 HLA-DPA1HLA-DPA1 0.75 0.75 0.63 0.63 0.03 0.03 HLA-DPA1HLA-DPA1 0.93 0.93 0.65 0.65 0.41 0.41 HLA-DPA1HLA-DPA1 0.78 0.78 0.54 0.54 0.18 0.18 SMOC2/THBS2SMOC2/THBS2 0.93 0.93 0.59 0.59 0.38 0.38 ADAP1ADAP1 0.98 0.98 0.49 0.49 0.33 0.33 ELFN1ELFN1 0.85 0.85 0.63 0.63 0.36 0.36 MAD1L1MAD1L1 0.90 0.90 0.52 0.52 0.31 0.31 LYPD2LYPD2 0.92 0.92 0.63 0.63 0.36 0.36 MAPK15MAPK15 0.84 0.84 0.52 0.52 0.33 0.33 PUF60PUF60 0.88 0.88 0.49 0.49 0.19 0.19 LINGO2/LINC01242LINGO2/LINC01242 0.93 0.93 0.62 0.62 0.40 0.40 APBA1APBA1 0.87 0.87 0.64 0.64 0.40 0.40 NCS1NCS1 0.87 0.87 0.49 0.49 0.30 0.30 FIBCD1FIBCD1 0.89 0.89 0.65 0.65 0.41 0.41 FIBCD1FIBCD1 0.91 0.91 0.60 0.60 0.38 0.38 FIBCD1FIBCD1 0.88 0.88 0.60 0.60 0.39 0.39 RXRA/COL5A1RXRA/COL5A1 0.91 0.91 0.59 0.59 0.31 0.31 KCNT1KCNT1 0.80 0.80 0.61 0.61 0.37 0.37 GPSM1GPSM1 0.96 0.96 0.56 0.56 0.33 0.33 PFKFB3PFKFB3 0.93 0.93 0.55 0.55 0.39 0.39 ATRNL1ATRNL1 0.86 0.86 0.63 0.63 0.41 0.41 FRG2B/DUX4L3FRG2B/DUX4L3 0.74 0.74 0.44 0.44 0.14 0.14 PKP3PKP3 0.87 0.87 0.31 0.31 0.14 0.14 MUC5BMUC5B 0.90 0.90 0.51 0.51 0.38 0.38 LMO1LMO1 0.91 0.91 0.57 0.57 0.37 0.37 LOC441601LOC441601 0.71 0.71 0.27 0.27 0.10 0.10 APOA5APOA5 0.91 0.91 0.51 0.51 0.39 0.39 TRIM29TRIM29 0.83 0.83 0.49 0.49 0.31 0.31 B4GALNT3B4GALNT3 0.94 0.94 0.51 0.51 0.36 0.36 CACNA2D4LRTM2CACNA2D4LRTM2 0.92 0.92 0.57 0.57 0.39 0.39 TSPAN9/PRMT8TSPAN9/PRMT8 0.76 0.76 0.47 0.47 0.27 0.27 VAMP1/MRPL51VAMP1/MRPL51 0.95 0.95 0.66 0.66 0.48 0.48 GNB3GNB3 0.90 0.90 0.58 0.58 0.39 0.39 LRIG3/SLC16A7LRIG3/SLC16A7 0.90 0.90 0.50 0.50 0.30 0.30 CABP1CABP1 0.92 0.92 0.52 0.52 0.35 0.35 TMEM132DTMEM132D 0.83 0.83 0.38 0.38 0.20 0.20 LINC01257LINC01257 0.85 0.85 0.55 0.55 0.35 0.35 LOC101928416/FBRSL1LOC101928416/FBRSL1 0.87 0.87 0.49 0.49 0.34 0.34 LINC00572LINC00572 0.93 0.93 0.43 0.43 0.28 0.28 MCF2LMCF2L 0.87 0.87 0.59 0.59 0.37 0.37 MCF2LMCF2L 0.94 0.94 0.62 0.62 0.47 0.47 GRTP1/ADPRHL1GRTP1/ADPRHL1 0.89 0.89 0.51 0.51 0.29 0.29 EAPP/SNX6EAPP/SNX6 0.91 0.91 0.56 0.56 0.35 0.35 ELK2AP/KIAA0125ELK2AP/KIAA0125 0.80 0.80 0.54 0.54 0.36 0.36 ADAM6/LINC00226ADAM6/LINC00226 0.90 0.90 0.46 0.46 0.17 0.17 LINC00226/LINC00221LINC00226/LINC00221 0.84 0.84 0.54 0.54 0.36 0.36 C15orf48C15orf48 0.87 0.87 0.60 0.60 0.33 0.33 SV2BSV2B 0.91 0.91 0.70 0.70 0.50 0.50 LOC101927332/FAM169BLOC101927332/FAM169B 0.85 0.85 0.50 0.50 0.32 0.32 SBK1SBK1 0.98 0.98 0.58 0.58 0.40 0.40 RRN3P2/SNX29P2RRN3P2/SNX29P2 0.91 0.91 0.35 0.35 0.17 0.17 ARHGEF15ARHGEF15 0.91 0.91 0.46 0.46 0.25 0.25 NFE2L1NFE2L1 0.96 0.96 0.54 0.54 0.33 0.33 SEC14L1/SEPT9SEC14L1/SEPT9 0.87 0.87 0.39 0.39 0.19 0.19 B3GNTL1/METRNLB3GNTL1/METRNL 0.93 0.93 0.31 0.31 0.14 0.14 AQP4-AS1AQP4-AS1 0.77 0.77 0.47 0.47 0.32 0.32 TSHZ1TSHZ1 0.89 0.89 0.53 0.53 0.36 0.36 GALR1/LINC01029GALR1/LINC01029 0.91 0.91 0.57 0.57 0.39 0.39 LINC01029, SALL3LINC01029, SALL3 0.86 0.86 0.57 0.57 0.36 0.36 GRIN3BGRIN3B 0.81 0.81 0.39 0.39 0.19 0.19 SMIM24/DOHHSMIM24/DOHH 0.84 0.84 0.56 0.56 0.34 0.34 C19orf67C19orf67 0.95 0.95 0.45 0.45 0.29 0.29 TMC4TMC4 0.90 0.90 0.55 0.55 0.37 0.37 LILRB3LILRB3 0.93 0.93 0.66 0.66 0.29 0.29 SIRPG/LOC100289473SIRPG/LOC100289473 0.94 0.94 0.43 0.43 0.27 0.27 FOXS1FOXS1 0.98 0.98 0.36 0.36 0.11 0.11 HELZ2HELZ2 0.95 0.95 0.57 0.57 0.37 0.37 LOC101927797/LINC00320LOC101927797/LINC00320 0.93 0.93 0.43 0.43 0.22 0.22 LINC00111LINC00111 0.92 0.92 0.58 0.58 0.42 0.42 TFF2TFF2 0.85 0.85 0.47 0.47 0.27 0.27 ZDHHC8P1ZDHHC8P1 0.89 0.89 0.55 0.55 0.38 0.38 LOC284930LOC284930 0.91 0.91 0.56 0.56 0.34 0.34

도 3, 도 4 및 표 6에 나타낸 바와 같이, LCK, FCRL5, RHOU/RAB4A, GREM2/RGS7, DCDC2C/LINC01304, RNU6-81P, CCDC108, LOC150935/MIR4786, AQP12B, AQP12A, LINC01237, XCR1, FGFR3, NDST4/MIR1973, TPPP, HNRNPH1/C5orf60, RASGEF1C, LINC01012/LOC100131289, HLA-DPA1, SMOC2/THBS2, ADAP1, ELFN1, MAD1L1, LYPD2, MAPK15, PUF60, LINGO2/LINC01242, APBA1, NCS1, FIBCD1, RXRA/COL5A1, KCNT1, GPSM1, PFKFB3, ATRNL1, FRG2B/DUX4L3, PKP3, MUC5B, LMO1, LOC441601, APOA5, TRIM29, B4GALNT3, CACNA2D4/LRTM2, TSPAN9/PRMT8, VAMP1/MRPL51, GNB3, LRIG3/SLC16A7, CABP1, TMEM132D, LINC01257, LOC101928416/FBRSL1, LINC00572, MCF2L, GRTP1/ADPRHL1, EAPP/SNX6, ELK2AP/KIAA0125, ADAM6/LINC00226, LINC00226/LINC00221, C15orf48, SV2B, LOC101927332/FAM169B, SBK1, RRN3P2/SNX29P2, ARHGEF15, NFE2L1, SEC14L1/SEPT9, B3GNTL1/METRNL, AQP4-AS1, TSHZ1, GALR1/LINC01029, LINC01029/SALL3, GRIN3B, SMIM24/DOHH, C19orf67, TMC4, LILRB3, SIRPG/LOC100289473, FOXS1, HELZ2, LOC101927797/LINC00320, LINC00111, TFF2, ZDHHC8P1 및 LOC284930 의 CpG 영역을 갖는 단편의 경우, 모체 혈액보다 정상 태아 태반에서 CpG 영역의 낮은 메틸화 수준을 나타냈고, 정상 태아 태반보다 에드워드 증후군 태아 태반에서 CpG 영역의 낮은 메틸화 수준을 나타냈다.As shown in FIG. 3, FIG. 4 and Table 6, LCK, FCRL5, RHOU/RAB4A, GREM2/RGS7, DCDC2C/LINC01304, RNU6-81P, CCDC108, LOC150935/MIR4786, AQP12B, AQP12A, LINC01237, XCR1, FGFR3, NDST4/MIR1973, TPPP, HNRNPH1/C5orf60, RASGEF1C, LINC01012/LOC100131289, HLA-DPA1, SMOC2/THBS2, ADAP1, ELFN1, MAD1L1, LYPD2, MAPK15, PUF60, LINGO2/LINC01242, APBA1, NCS1, FIBCD1, RXRA/COL5A1, KCNT1, GPSM1, PFKFB3, ATRNL1, FRG2B/DUX4L3, PKP3, MUC5B, LMO1, LOC441601, APOA5, TRIM29, B4GALNT3, CACNA2D4/LRTM2, TSPAN9/PRMT8, VAMP1/MRPL51, GNB3, LRIG3/SLC16A7, CABP1, TMEM132D, LINC01257, LOC101928416/FBRSL1, LINC00572, MCF2L, GRTP1/ADPRHL1, EAPP/SNX6, ELK2AP/KIAA0125, ADAM6/LINC00226, LINC00226/LINC00221, C15orf48, SV2B, For fragments with CpG regions of LOC101927332/FAM169B, SBK1, RRN3P2/SNX29P2, ARHGEF15, NFE2L1, SEC14L1/SEPT9, B3GNTL1/METRNL, AQP4-AS1, TSHZ1, GALR1/LINC01029, LINC01029/SALL3, GRIN3B, SMIM24/DOHH, C19orf67, TMC4, LILRB3, SIRPG/LOC100289473, FOXS1, HELZ2, LOC101927797/LINC00320, LINC00111, TFF2, ZDHHC8P1 , and LOC284930, lower methylation levels of CpG regions were observed in normal fetal placentas than in maternal blood, and normal Lower methylation levels of CpG regions were observed in Edwards syndrome placentas compared to placentas of control fetuses.

상기 91개의 유전자(또는 이의 단편)을 저메틸화 바이오마커로 선정하였으며, 상기 바이오마커에 있어서, CpG site의 메틸화 수준이 정상 태아 태반이 모체 혈액보다 50 내지 75 정도 낮았으며, 에드워드 증후군 태아 태반이 정상 태아 태반 보다 10 내지 50 정도 낮았다.The above 91 genes (or fragments thereof) were selected as hypomethylated biomarkers, and the methylation level of the CpG site in the above biomarkers was about 50 to 75 lower in the normal fetal placenta than in the maternal blood, and about 10 to 50 lower in the Edwards syndrome fetal placenta than in the normal fetal placenta.

상기 바이오마커에 있어서, 에드워드 증후군 태아 태반과 다른 두 그룹 간의 메틸화 수준의 차이는 모두 통계적으로 유의하였다(p value < 0.05).For the above biomarkers, the differences in methylation levels between the Edwards syndrome fetal placentas and the other two groups were all statistically significant (p value < 0.05).

Claims (8)

태반으로부터 분리된, CpG 영역을 갖는 유전자의 CpG 영역의 메틸화 수준을 측정하는 제제를 포함하는, 에드워드 증후군 진단용 조성물.A composition for diagnosing Edwards syndrome, comprising a preparation for measuring the methylation level of a CpG region of a gene having a CpG region, isolated from the placenta. 청구항 1에 있어서, 상기 유전자는 하기 (a) 및 (b)로 이루어진 군에서 선택되는 하나 이상의 유전자를 포함하는 것인, 에드워드 증후군 진단용 조성물:A composition for diagnosing Edwards syndrome according to claim 1, wherein the gene comprises at least one gene selected from the group consisting of (a) and (b): (a) MXRA8, PTPRU, IL12RB2, WNT3A, KIF26B, SLC30A3/DNAJC5G, ZFP36L2, ZIC4, CPLX1, ZFYVE28, CCNG2/CXCL13, PALLD, IRX4, C5orf38, ISL1, LHFPL2, GPRIN1, LOC100506207/TFAP2A, GFOD1/SIRT5, ETV7, FOXP4, SMOC2, GNA12/CARD11, AP5Z1, ZNF273/ZNF117, CALN1, SND1, HTR5A/INSIG1, MNX1, EBF2/PPP2R2A, FAM110B, OSR2/VPS13B, NAPRT, GLIS3, SLC24A2/MLLT3, CRB2, RABL6, TACC2, CPXM2/CHST15, CLMP, KRT7/KRT81, HOXC4, LOC100287944, LINC00403, LINC00925, NR2F2-AS1, MGRN1, RBFOX1, PLEKHH3, EMILIN2, ST8SIA3, ST8SIA3/ONECUT2, ONECUT2, NFATC1/CTDP1, PARD6G, ZNF563, MED29/ZFP36, DYRK1B, PPP1R13L, CYP24A1, C21orf2/TRPM2, YBEY, MAPK8IP2 및 이들의 CpG 영역을 갖는 단편; 및 (a) MXRA8, PTPRU, IL12RB2, WNT3A, KIF26B, SLC30A3/DNAJC5G, ZFP36L2, ZIC4, CPLX1, ZFYVE28, CCNG2/CXCL13, PALLD, IRX4, C5orf38, ISL1, LHFPL2, GPRIN1, LOC100506207/TFAP2A, GFOD1/SIRT5, ETV7, FOXP4, SMOC2, GNA12/CARD11, AP5Z1, ZNF273/ZNF117, CALN1, SND1, HTR5A/INSIG1, MNX1, EBF2/PPP2R2A, FAM110B, OSR2/VPS13B, NAPRT, GLIS3, SLC24A2/MLLT3, CRB2, RABL6, TACC2, CPXM2/CHST15, CLMP, KRT7/KRT81, HOXC4, LOC100287944, LINC00403, LINC00925, NR2F2-AS1, MGRN1, RBFOX1, PLEKHH3, EMILIN2, ST8SIA3, ST8SIA3/ONECUT2, ONECUT2, NFATC1/CTDP1, PARD6G, ZNF563, MED29/ZFP36, DYRK1B, PPP1R13L, CYP24A1, C21orf2/TRPM2, YBEY , MAPK8IP2 and fragments having their CpG regions; and (b) LCK, FCRL5, RHOU/RAB4A, GREM2/RGS7, DCDC2C/LINC01304, RNU6-81P, CCDC108, LOC150935/MIR4786, AQP12B, AQP12A, LINC01237, XCR1, FGFR3, NDST4/MIR1973, TPPP, HNRNPH1/C5orf60, RASGEF1C, LINC01012/LOC100131289, HLA-DPA1, SMOC2/THBS2, ADAP1, ELFN1, MAD1L1, LYPD2, MAPK15, PUF60, LINGO2/LINC01242, APBA1, NCS1, FIBCD1, RXRA/COL5A1, KCNT1, GPSM1, PFKFB3, ATRNL1, FRG2B/DUX4L3, PKP3, MUC5B, LMO1, LOC441601, APOA5, TRIM29, B4GALNT3, CACNA2D4/LRTM2, TSPAN9/PRMT8, VAMP1/MRPL51, GNB3, LRIG3/SLC16A7, CABP1, TMEM132D, LINC01257, LOC101928416/FBRSL1, LINC00572, MCF2L, GRTP1/ADPRHL1, EAPP/SNX6, ELK2AP/KIAA0125, ADAM6/LINC00226, LINC00226/LINC00221, C15orf48, SV2B, LOC101927332/FAM169B, SBK1, RRN3P2/SNX29P2, ARHGEF15, NFE2L1, SEC14L1/SEPT9, B3GNTL1/METRNL, AQP4-AS1, TSHZ1, GALR1/LINC01029, LINC01029/SALL3, GRIN3B, SMIM24/DOHH, C19orf67, TMC4, LILRB3, SIRPG/LOC100289473, FOXS1, HELZ2, LOC101927797/LINC00320, LINC00111, TFF2, ZDHHC8P1 LOC284930 및 이들의 CpG 영역을 갖는 단편.(b) LCK, FCRL5, RHOU/RAB4A, GREM2/RGS7, DCDC2C/LINC01304, RNU6-81P, CCDC108, LOC150935/MIR4786, AQP12B, AQP12A, LINC01237, XCR1, FGFR3, NDST4/MIR1973, TPPP, HNRNPH1/C5orf60, RASGEF1C, LINC01012/LOC100131289, HLA-DPA1, SMOC2/THBS2, ADAP1, ELFN1, MAD1L1, LYPD2, MAPK15, PUF60, LINGO2/LINC01242, APBA1, NCS1, FIBCD1, RXRA/COL5A1, KCNT1, GPSM1, PFKFB3, ATRNL1, FRG2B/DUX4L3, PKP3, MUC5B, LMO1, LOC441601, APOA5, TRIM29, B4GALNT3, CACNA2D4/LRTM2, TSPAN9/PRMT8, VAMP1/MRPL51, GNB3, LRIG3/SLC16A7, CABP1, TMEM132D, LINC01257, LOC101928416/FBRSL1, LINC00572, MCF2L, GRTP1/ADPRHL1, EAPP/SNX6, ELK2AP/KIAA0125, ADAM6/LINC00226, LINC00226/LINC00221, C15orf48, SV2B, LOC101927332/FAM169B, SBK1, RRN3P2/SNX29P2, ARHGEF15, NFE2L1, SEC14L1/SEPT9, B3GNTL1/METRNL, AQP4-AS1, TSHZ1, GALR1/LINC01029, LINC01029/SALL3, GRIN3B, SMIM24/DOHH, C19orf67, TMC4, LILRB3, SIRPG/LOC100289473, FOXS1, HELZ2, LOC101927797/LINC00320, LINC00111, TFF2, ZDHHC8P1 LOC284930 and fragments having their CpG regions. 제1항에 있어서, 상기 유전자의 CpG 영역의 메틸화 수준을 측정하는 제제는, 바이설파이트(bisulfite) 또는 이의 염, 메틸화 민감성 제한효소, 상기 유전자의 CpG 부위를 포함하는 서열에 특이적으로 결합하는 프라이머, 상기 유전자의 메틸화된 CpG 부위를 포함하는 서열에 혼성화할 수 있는 프로브, 메틸화된 CpG 결합 도메인, 또는 메틸사이토신에 특이적으로 결합하는 항체 또는 압타머를 포함하는, 에드워드 증후군 진단용 조성물.In the first paragraph, a composition for diagnosing Edwards syndrome, wherein the agent for measuring the methylation level of the CpG region of the gene comprises bisulfite or a salt thereof, a methylation-sensitive restriction enzyme, a primer that specifically binds to a sequence including a CpG site of the gene, a probe that can hybridize to a sequence including a methylated CpG site of the gene, a methylated CpG binding domain, or an antibody or aptamer that specifically binds to methylcytosine. 청구항 1 내지 3 중 어느 한 항의 조성물을 포함하는, 에드워드 증후군 진단용 키트.A kit for diagnosing Edwards syndrome, comprising a composition according to any one of claims 1 to 3. 대상체로부터 분리된 태반시료에서 CpG 영역을 갖는 유전자의 CpG 영역의 메틸화 수준을 측정하는 단계; 및 상기 단계에서 측정된 메틸화 수준을 모체 혈액 및 정상 태반 내 동일 유전자의 CpG 영역의 메틸화 수준과 비교하는 단계를 포함하는, 에드워드 증후군의 진단을 위한 정보를 제공하는 방법.A method for providing information for diagnosing Edwards syndrome, comprising: a step of measuring a methylation level of a CpG region of a gene having a CpG region in a placental sample isolated from a subject; and a step of comparing the methylation level measured in the step with the methylation level of the CpG region of the same gene in maternal blood and a normal placenta. 청구항 5에 있어서, 상기 메틸화 수준을 측정하는 단계는 PCR, 메틸화 특이적 PCR(methylation-specific polymerase chain reaction), 실시간 메틸화 특이적 PCR(real time methylation-specific polymerase chain reaction), MethyLight PCR, MehtyLight digital PCR, EpiTYPER, 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 메틸 PNA-PCR, 정량 PCR, DNA 칩, 파이로시퀀싱, 바이설파이트 시퀀싱, 서던블롯, RLGS(Restriction landmark genomic scanning), MS-SnuPE(Methylation-sensitive single-nucleotide primer extension), CpG 마이크로어레이, COBRA(Combined bisulfite-restriction analysis), MIRA(methylated-CpG island recovery assay), 질량 스펙트럼법, 및 메틸화된 CpG 결합 도메인 또는 항-메틸사이토신 항체를 이용한 면역침강법으로 이루어진 군으로부터 선택되는 어느 하나 이상에 의해 수행되는 것인, 방법.A method according to claim 5, wherein the step of measuring the methylation level is performed by at least one selected from the group consisting of PCR, methylation-specific PCR, real time methylation-specific PCR, MethyLight PCR, MehtyLight digital PCR, EpiTYPER, PCR using methylated DNA-specific binding protein, methyl PNA-PCR, quantitative PCR, DNA chip, pyrosequencing, bisulfite sequencing, Southern blot, RLGS (Restriction landmark genomic scanning), MS-SnuPE (Methylation-sensitive single-nucleotide primer extension), CpG microarray, COBRA (Combined bisulfite-restriction analysis), MIRA (methylated-CpG island recovery assay), mass spectrometry, and immunoprecipitation using a methylated CpG binding domain or an anti-methylcytosine antibody. 청구항 5에 있어서, 상기 유전자는 하기 (a) 및 (b)로 이루어진 군에서 선택되는 하나 이상의 유전자를 포함하는 것인, 방법:A method according to claim 5, wherein the gene comprises at least one gene selected from the group consisting of (a) and (b): (a) MXRA8, PTPRU, IL12RB2, WNT3A, KIF26B,SLC30A3/DNAJC5G, ZFP36L2, ZIC4, CPLX1, ZFYVE28, CCNG2/CXCL13, PALLD, IRX4, C5orf38, ISL1, LHFPL2, GPRIN1, LOC100506207/TFAP2A, GFOD1/SIRT5, ETV7, FOXP4, SMOC2, GNA12/CARD11, AP5Z1, ZNF273/ZNF117, CALN1, SND1, HTR5A/INSIG1, MNX1, EBF2/PPP2R2A, FAM110B, OSR2/VPS13B, NAPRT, GLIS3, SLC24A2/MLLT3, CRB2, RABL6, TACC2, CPXM2/CHST15, CLMP, KRT7/KRT81, HOXC4, LOC100287944, LINC00403, LINC00925, NR2F2-AS1, MGRN1, RBFOX1, PLEKHH3, EMILIN2, ST8SIA3, ST8SIA3/ONECUT2, ONECUT2, NFATC1/CTDP1, PARD6G, ZNF563, MED29/ZFP36, DYRK1B, PPP1R13L, CYP24A1, C21orf2/TRPM2, YBEY, MAPK8IP2 및 이들의 CpG 영역을 갖는 단편; 및 (a) MXRA8, PTPRU, IL12RB2, WNT3A, KIF26B, SLC30A3/DNAJC5G, ZFP36L2, ZIC4, CPLX1, ZFYVE28, CCNG2/CXCL13, PALLD, IRX4, C5orf38, ISL1, LHFPL2, GPRIN1, LOC100506207/TFAP2A, GFOD1/SIRT5, ETV7, FOXP4, SMOC2, GNA12/CARD11, AP5Z1, ZNF273/ZNF117, CALN1, SND1, HTR5A/INSIG1, MNX1, EBF2/PPP2R2A, FAM110B, OSR2/VPS13B, NAPRT, GLIS3, SLC24A2/MLLT3, CRB2, RABL6, TACC2, CPXM2/CHST15, CLMP, KRT7/KRT81, HOXC4, LOC100287944, LINC00403, LINC00925, NR2F2-AS1, MGRN1, RBFOX1, PLEKHH3, EMILIN2, ST8SIA3, ST8SIA3/ONECUT2, ONECUT2, NFATC1/CTDP1, PARD6G, ZNF563, MED29/ZFP36, DYRK1B, PPP1R13L, CYP24A1, C21orf2/TRPM2, YBEY, MAPK8IP2 and fragments having their CpG regions; and (b) LCK, FCRL5, RHOU/RAB4A, GREM2/RGS7, DCDC2C/LINC01304, RNU6-81P, CCDC108, LOC150935/MIR4786, AQP12B, AQP12A, LINC01237, XCR1, FGFR3, NDST4/MIR1973, TPPP, HNRNPH1/C5orf60, RASGEF1C, LINC01012/LOC100131289, HLA-DPA1, SMOC2/THBS2, ADAP1, ELFN1, MAD1L1, LYPD2, MAPK15, PUF60, LINGO2/LINC01242, APBA1, NCS1, FIBCD1, RXRA/COL5A1, KCNT1, GPSM1, PFKFB3, ATRNL1, FRG2B/DUX4L3, PKP3, MUC5B, LMO1, LOC441601, APOA5, TRIM29, B4GALNT3, CACNA2D4/LRTM2, TSPAN9/PRMT8, VAMP1/MRPL51, GNB3, LRIG3/SLC16A7, CABP1, TMEM132D, LINC01257, LOC101928416/FBRSL1, LINC00572, MCF2L, GRTP1/ADPRHL1, EAPP/SNX6, ELK2AP/KIAA0125, ADAM6/LINC00226, LINC00226/LINC00221, C15orf48, SV2B, LOC101927332/FAM169B, SBK1, RRN3P2/SNX29P2, ARHGEF15, NFE2L1, SEC14L1/SEPT9, B3GNTL1/METRNL, AQP4-AS1, TSHZ1, GALR1/LINC01029, LINC01029/SALL3, GRIN3B, SMIM24/DOHH, C19orf67, TMC4, LILRB3, SIRPG/LOC100289473, FOXS1, HELZ2, LOC101927797/LINC00320, LINC00111, TFF2, ZDHHC8P1, LOC284930 및 이들의 CpG 영역을 갖는 단편. (b) LCK, FCRL5, RHOU/RAB4A, GREM2/RGS7, DCDC2C/LINC01304, RNU6-81P, CCDC108, LOC150935/MIR4786, AQP12B, AQP12A, LINC01237, XCR1, FGFR3, NDST4/MIR1973, TPPP, HNRNPH1/C5orf60, RASGEF1C, LINC01012/LOC100131289, HLA-DPA1, SMOC2/THBS2, ADAP1, ELFN1, MAD1L1, LYPD2, MAPK15, PUF60, LINGO2/LINC01242, APBA1, NCS1, FIBCD1, RXRA/COL5A1, KCNT1, GPSM1, PFKFB3, ATRNL1, FRG2B/DUX4L3, PKP3, MUC5B, LMO1, LOC441601, APOA5, TRIM29, B4GALNT3, CACNA2D4/LRTM2, TSPAN9/PRMT8, VAMP1/MRPL51, GNB3, LRIG3/SLC16A7, CABP1, TMEM132D, LINC01257, LOC101928416/FBRSL1, LINC00572, MCF2L, GRTP1/ADPRHL1, EAPP/SNX6, ELK2AP/KIAA0125, ADAM6/LINC00226, LINC00226/LINC00221, C15orf48, SV2B, LOC101927332/FAM169B, SBK1, RRN3P2/SNX29P2, ARHGEF15, NFE2L1, SEC14L1/SEPT9, B3GNTL1/METRNL, AQP4-AS1, TSHZ1, GALR1/LINC01029, LINC01029/SALL3, GRIN3B, SMIM24/DOHH, C19orf67, TMC4, LILRB3, SIRPG/LOC100289473, FOXS1, HELZ2, LOC101927797/LINC00320, LINC00111, TFF2, ZDHHC8P1, LOC284930 and fragments having their CpG regions . 청구항 7에 있어서, 상기 (a)의 유전자의 메틸화 수준이 모체 혈액 및 정상 태반 내 동일 유전자의 CpG 영역의 메틸화 수준에 비해 증가된 경우, 에드워드 증후군으로 판단하거나,In claim 7, if the methylation level of the gene of (a) is increased compared to the methylation level of the CpG region of the same gene in maternal blood and normal placenta, it is determined to be Edwards syndrome, or 상기 (b)의 유전자의 메틸화 수준이 모체 혈액 및 정상 태반 내 동일 유전자의 CpG 영역의 메틸화 수준에 비해 감소된 경우, 에드워드 증후군으로 판단하는 단계를 더 포함하는 것인, 방법.A method further comprising a step of determining Edwards syndrome when the methylation level of the gene of the above (b) is decreased compared to the methylation level of the CpG region of the same gene in maternal blood and normal placenta.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160340740A1 (en) * 2014-01-30 2016-11-24 The Regents Of The University Of California Methylation haplotyping for non-invasive diagnosis (monod)
KR102036609B1 (en) * 2018-02-12 2019-10-28 바이오코아 주식회사 A method for prenatal diagnosis using digital PCR
KR20200028153A (en) * 2018-09-06 2020-03-16 이원다이애그노믹스(주) Non-invasive prenatal testing method using cell free dna fragment derived maternal blood

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100804419B1 (en) 2007-05-28 2008-02-20 주식회사 마크로젠 Microarray and kit for diagnosing edwards syndrome

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160340740A1 (en) * 2014-01-30 2016-11-24 The Regents Of The University Of California Methylation haplotyping for non-invasive diagnosis (monod)
KR102036609B1 (en) * 2018-02-12 2019-10-28 바이오코아 주식회사 A method for prenatal diagnosis using digital PCR
KR20200028153A (en) * 2018-09-06 2020-03-16 이원다이애그노믹스(주) Non-invasive prenatal testing method using cell free dna fragment derived maternal blood

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DANA W. Y. TSUI, Y. M. DORIS LAM, WING S. LEE, TAK Y. LEUNG, TZE K. LAU, ELIZABETH T. LAU, MARY H. Y. TANG, RANJIT AKOLEKAR, KYPRO: "Systematic Identification of Placental Epigenetic Signatures for the Noninvasive Prenatal Detection of Edwards Syndrome", PLOS ONE, vol. 5, no. 11, pages e15069, XP055083773, DOI: 10.1371/journal.pone.0015069 *
HATT LOTTE, AAGAARD MADS M., BACH CATHRINE, GRAAKJAER JESPER, SOMMER STEFFEN, AGERHOLM INGE E., KØLVRAA STEEN, BOJESEN ANDERS: "Microarray-Based Analysis of Methylation of 1st Trimester Trisomic Placentas from Down Syndrome, Edwards Syndrome and Patau Syndrome", PLOS ONE, vol. 11, no. 8, US , pages e0160319 - e0160319-11, XP093290505, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0160319 *

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