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

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

Disease-specific epigenetic biomarkers for diagnosing Edwards syndrome and their uses

[0001] Disease-specific epigenetic biomarkers for diagnosing Edwards syndrome and their uses.

[0002] 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 problems, and muscle weakness. More than 90% of cases die within 6 months of birth, and only about 5% survive to 1 year of age.

[0003] Due to factors such as the aging marriage population and the increasing age of childbirth, the risk of developing genetic diseases in the fetus has been rapidly increasing over the past decade. Early detection of diseases involving genetic abnormalities in the fetus early in pregnancy allows for prompt medical intervention to ensure safety while minimizing suffering for both the mother and fetus. Therefore, early diagnosis of genetic abnormalities in the fetus is crucial.

[0004] Currently, for Edwards syndrome, a prenatal diagnosis method (NGS-based NIPT) that analyzes quantitative differences in cfDNA, a non-cellular DNA fragment present in maternal plasma, has been developed and is rapidly being applied clinically. However, NGS-based NIPT requires complex analytical methods, making it difficult to apply in routine laboratories. Furthermore, it requires expensive analytical equipment and consumables, leading to high testing costs. This limits its application to all pregnant women.

[0005] Another prenatal diagnosis method for Edwards syndrome, PCR-based NIPT, uses SNPs for analysis, so there is no clear basis for its accuracy. In addition, it requires the use of special algorithms presented by various institutions, which limits its general application.

[0006] Accordingly, there is a need for a method that can effectively diagnose Edwards syndrome while solving the problems of conventional technology.

[0007] 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 equipment or special algorithms.

[0008] 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 the placenta.

[0009] 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.

[0010] Another aspect provides a use for preparing 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.

[0011] Another aspect is to provide a kit for diagnosing Edwards syndrome, comprising the composition.

[0012] 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.

[0013] Another aspect provides a method for diagnosing Edwards syndrome, comprising the steps of: measuring the 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 a CpG region of the same gene in maternal blood and a normal placenta.

[0014] 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 the placenta.

[0015] Another aspect provides the use of a preparation for diagnosing Edwards syndrome by measuring the methylation level of a CpG region of a gene having a CpG region, isolated from the placenta.

[0016] 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 the placenta.

[0017] Another aspect provides the use of genes with CpG regions isolated from the placenta for the diagnosis of Edwards syndrome.

[0018] The term "placenta" as used herein refers to an organ that connects the fetus and the mother's endometrium, and is responsible for functions such as supplying oxygen and nutrients from the mother, protecting the fetus, and excreting fetal waste. It may include the placenta and umbilical cord blood. In addition, the placenta may not refer to the placenta discharged after birth, but rather to a chorionic villus sample used in CVS (Chorionic Villi Sampling), an early prenatal test.

[0019] As used herein, the term "CpG region" refers to a DNA region of a gene or a portion thereof where CpGs are concentrated, called a CpG island. For example, the CpG region may be present in a transcriptional regulatory region such as a promoter region, a protein coding region (open reading frame, ORF), or a terminator region.

[0020] As used herein, the term "methylation" 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 at a cytosine in a specific CpG region of a specific gene. If methylation occurs, it may inhibit the binding of transcription factors, thereby suppressing the expression of the corresponding gene. The suppression of expression 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 regulating gene expression. It is a methyl group (-CH3) attached to the 5th carbon of cytosine, transforming it into the form of 5-methylcytosine. DNA methylation primarily occurs at the cytosine group of CpG dinucleotides and is prevalent in regions of DNA where CpG is concentrated, called CpG islands or CpG tracts.

[0021] In one specific example, the gene may comprise one or more genes selected from the group consisting of (a) and (b):

[0022] (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

[0023] <h2 style=";text-align:left;direction:ltr">(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, LOC284930more 영역을 갖는 단편.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0024] In one specific example, the gene of (a) may be selected from Table 1 below.

[0025]

[0026]

[0027] In one specific example, the gene of (b) may be selected from Table 2 below.

[0028]

[0029]

[0030] In this specification, "gene A / gene B" means a region existing between two genes, and specifically means a region between two genes excluding the region 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 genes A and B.

[0031] In this specification, a fragment containing the CpG region of a gene may refer to a fragment that has selectivity and specificity for the gene, such that it can represent the gene among fragments containing the CpG region of the gene. Accordingly, by measuring the methylation level of a fragment containing the CpG region of any gene, the methylation level of the gene can be compared, thereby diagnosing Edwards syndrome.

[0032] Furthermore, 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 containing a CpG region within the gene to achieve the desired effect of the present invention, or utilize a fragment containing a CpG region known to represent any gene in the art. Furthermore, agents that measure the methylation level of such fragments can be purchased and compared with the methylation level of the desired gene.

[0033]

[0034] 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 the epigenetic characteristic is maintained, as 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, normal fetus, and Edwards syndrome fetus exist.

[0035] 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.

[0036] 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.

[0037] Additionally, the measurement of the methylation level of the above CpG region may be measured at the genome level.

[0038] The term "marker" or "biomarker" in this specification means 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 an immune-related disease.

[0039] 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 with a particular disease or condition, or monitoring the same.

[0040] 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.

[0041] The above Edwards syndrome may have congenital external malformations, a small and narrow head, protruding occipital bone, microstomia, cleft lip and palate, micrognathia, short sternum, small pelvis, characteristic hand and foot appearance (index finger over middle finger and little finger over ring finger), rocker-bottom feet, short and backward-bent first toes, developmental delay, mental retardation, severe mental retardation, cardiac malformations (ventricular septal defect, patent ductus arteriosus), renal malformations (horse's knee), diaphragmatic hernia, tracheoesophageal fistula, respiratory problems, and muscle weakness.

[0042] 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.

[0043]

[0044] Another aspect provides a kit for diagnosing Edwards syndrome, comprising the composition.

[0045] The Edwards syndrome is as described above.

[0046] 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 as described above or a fragment thereof; (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.

[0047] The 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.

[0048] Specifically, the kit may be comprised of one or more different component compositions, solutions, or devices suitable for the analytical method. For example, the kit may be an RT-PCR (Reverse Transcription Polymerase Chain Reaction) kit or a DNA chip kit. In addition to the above formulation, the kit may additionally include polymerase agarose, a buffer solution required for electrophoresis, and the like.

[0049] In one specific example, the kit may include a diagnostic nucleic acid chip (CHIP) or gene panel having a probe immobilized thereon that can hybridize with a gene having the CpG region or a fragment thereof having the CpG region.

[0050] In one specific example, the kit may include a preparation, a device, and a computer having an algorithm built in 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.

[0051]

[0052] 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.

[0053] 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.

[0054] 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.

[0055] The placenta, CpG region, level measurement, methylation level measurement of the CpG region, diagnosis and Edwards syndrome are as described above.

[0056] 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 (methylation-specific polymerase chain reaction), real-time methylation-specific PCR (real-time methylation-specific polymerase chain reaction), 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 spectroscopy, and immunoprecipitation using a methylated CpG binding domain or an anti-methylcytosine antibody.

[0057] In one specific example, the gene may comprise one or more genes selected from the group consisting of (a) and (b):

[0058] (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

[0059] <h2 style=";text-align:left;direction:ltr">(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, LOC284930more 영역을 갖는 단편.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0060] In one specific example, the gene of (a) may be selected from Table 1 above.

[0061] In one specific example, the gene of (b) may be selected from Table 2 above.

[0062] 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.

[0063] As used herein, the term “increased methylation level of a gene” or “increased methylation level of a CpG region of a gene” means that the methylation level in a sample of a subject is measurably and significantly increased compared to a control group, and may mean, for example, an increase of about 1.1 times or more, for example, 1.1 to 2.5 times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, or 2 times or more.

[0064] As used herein, the term “decreased methylation level of a gene” or “decreased methylation level of a CpG region of a gene” means that the methylation level in a sample of a subject is measurably and significantly reduced compared to a control group, for example, by about 0.9 times or less, for example, by 0.1 times to 0.9 times, 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, 0.4 times, 0.3 times, 0.2 times, or 0.1 times or less.

[0065] In one specific example, the method for diagnosing Edwards syndrome can be performed using a plurality of panels.

[0066] A composition for diagnosing Edwards syndrome according to an 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.

[0067] Figures 1 and 2 show the results of a heat map analysis of the levels of Edwards syndrome-specific hypermethylation biomarkers selected for Edwards syndrome diagnosis.

[0068] Figures 3 and 4 show the results of a heat map analysis of the levels of Edwards syndrome-specific hypomethylated biomarkers selected for Edwards syndrome diagnosis.

[0069] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0070] 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 to explain his or her own invention in the best way.

[0071] Throughout the specification of the present invention, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0072] Throughout the specification of the present invention, “A and / or B” means A or B, or A and B.

[0073]

[0074] Example 1. Blood genome extraction

[0075] Genomes were extracted from blood samples from five maternal cases.

[0076] The DNeasy Blood & Tissue mini kits from Qiagen were used according to the manufacturer's instructions, and the detailed method is 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. 100% ethanol was then 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. 500 ml of Buffer AW1 was added for purification, centrifuged, and then 500 μl of Buffer AW2 was added, centrifuged again, and the purified sample was recovered using the DNeasy Mini spin column, and 50 μl of Buffer AE was added to base-bind the DNA and then recovered. Finally, the purification steps were repeated to complete the work so that the total volume became 100 μl.

[0077]

[0078] Example 2. Placental genome extraction

[0079] Genomes were extracted from placental samples from five normal fetuses and five fetuses with Edwards syndrome.

[0080] The tissue was placed in a 1.5 ml microtube (Axygen), and 3 mm tungsten carbide beads, 20 μl of proteinase K, and 200 μl of ATL buffer were added. The Tissuelyzer was operated at 25 Hz for 3 minutes. Incubation was then performed at 56°C for 20 minutes. Subsequent procedures were performed using Qiagen's DNeasy Blood & Tissue mini kits according to the manufacturer's instructions, as detailed below. After adding 20 μl of RNase and 200 μl of AL buffer, the tube was thoroughly mixed. 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.

[0081]

[0082] Example 3. Selection of biomarkers for diagnosing Edwards syndrome

[0083] To select biomarkers for diagnosing Edwards syndrome, methyl capture sequencing (MC-seq) analysis was performed after blood or placental genome extraction.

[0084] The blood and placental genomes extracted in Examples 1 and 2 were analyzed using MC-seq.

[0085] Specifically, the methylation level of the CpG region of a gene or fragment thereof having a CpG region was analyzed using MC-seq.

[0086] To select biomarkers for Edwards syndrome diagnosis, genomic DNA integrity of 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).

[0087] The process is as follows. 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 DNA methylated adapters are quantitatively quantified using the TapeStation DNA screentape D1000 (Agilent). For MC-seq, 350 ng of the 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 in a PCR machine using a thermal cycler lid option heated at 105°C. 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). Subsequently, they are sequenced using the NovaSeq platform (Illumina, San Diego, USA).

[0088] The data processing and methylation calling methods generated thereafter 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, bases that do not fit the window size = 4 and the average quality = 15 are removed using the sliding window trimming method, and reads with a minimum length of 36 bp are removed to generate cleaned data. The cleaned reads are aligned to the human genome (UCSC hg19) using BSMAP. BSMAP (version 2.90, parameter settings -n 1 -r 0) only accepts uniquely mapped reads, and the mapped data (SAM file format) are aligned and indexed using sambamba (version 0.6.5). Afterwards, 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).

[0089] In analysis, the methylation level of a CpG site is expressed as 0 to 100, with 0 indicating unmethylation and 100 indicating complete methylation.

[0090] In selecting biomarkers, we identified hypermethylated and hypomethylated markers based on epigenetic characteristics observed in the placenta of Edwards syndrome fetuses. Regions with two or more consecutive epigenetic characteristics, including the marker region, were selected. Specifically, genes or fragments thereof with regions where the methylation level of CpG regions increased or decreased sequentially in the order of the mother, normal fetus, and Edwards syndrome fetus were selected as biomarkers.

[0091]

[0092] 3.1. Selection of hypermethylation biomarkers for the diagnosis of Edwards syndrome

[0093] Genes or fragments thereof with higher methylation levels in normal fetal placentas than in maternal blood and with higher methylation levels in Edwards syndrome fetal placentas than in normal fetal blood were selected as hypermethylation biomarkers. Specifically, the selected hypermethylation biomarkers show a low methylation level of 0.2 or less in maternal blood, a high hypermethylation level of △ + 0.2 to + 0.3 in normal fetuses compared to maternal blood, and a high hypermethylation level of △ + 0.2 to + 0.3 in Edwards syndrome fetuses compared to normal fetuses.

[0094] The 67 selected hypermethylation biomarkers are shown in Table 3 below.

[0095]

[0096]

[0097]

[0098] 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.

[0099] Figures 1 and 2 show the results of a heat map analysis of the levels of Edwards syndrome-specific hypermethylation biomarkers selected for Edwards syndrome diagnosis.

[0100] In the above figure, NB represents maternal blood, NT represents the placenta of a normal fetus, and T18T represents the placenta of a fetus with Edwards syndrome.

[0101]

[0102] Gene methylation levelNormal maternal bloodNormal fetal placentaEdwards syndrome fetal placentaMXRA80.150.480.67PTPRU0.020.270.49IL12RB20.020.280.52WNT3A0.020.090.39KIF26B0.130.370.56SLC30A3 / DNAJC5G0.020.200.44ZFP36L20.030.330.56ZIC40.020.340.61CPLX10.040.300.56ZFYVE280.160.470.64CCNG2 / CXCL130.020.390.57PALLD0.010.330.56IRX40.100.49 0.72C5orf380.040.290.48ISL10.040.360.52LHFPL20.020.280.45GPRIN10.020.200.43LOC100506207 / TFAP2A0.120.270.44GFOD1 / SIRT50.1 80.430.65ETV70.090.290.49FOXP40.010.330.52SMOC20.040.310.57GNA12 / CARD110.020.420.58AP5Z10.100.560.78ZNF273 / ZNF1170.030.52 0.71CALN10.010.260.48SND10.010.260.55HTR5A / INSIG10.030.340.52MNX10.010.310.51EBF2 / PPP2R2A0.020.380.57FAM110B0.020.340.56 OSR2 / VPS13B0.080.560.72NAPRT0.040.180.55GLIS30.010.350.58SLC24A2 / MLLT30.090.360.53CRB20.090.440.62RABL60.240.500.73TACC20 .020.300.56CPXM2 / CHST150.060.380.64CLMP0.060.340.55KRT7 / KRT810.020.360.56HOXC40.030.430.60LOC1002879440.070.280.50LINC004 030.080.400.56LINC004030.060.360.55LINC009250.140.430.56NR2F2-AS10.240.550.68MGRN10.170.580.70RBFOX10.060.290.46PLEKHH30.120.610.87EMILIN20.060.300.50ST8SIA30.060.300.49ST8SIA3 / ONECUT20.050.330.56ST8SIA3 / ONECUT20.080.490.64ST8SIA3 / ONECUT20.080.500.67ONECUT20.030.320.49NFATC1 / CTDP10.100.470.59PARD6G0.290.560.78ZNF5630.010.510.73MED29 / ZFP360.030.350.52DYRK1B0.010.340.53PPP1R13L0.090.420.61CYP24A10.020.330.49CYP24A10.020.340.48C21orf2 / TRPM20.030.240.46YBEY0.090.480.63MAPK8IP20.030.280.49.

[0103]

[0104] As shown in Figure 1, Figure 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 placentas than in maternal blood, and in Edwards syndrome than in normal fetal placentas. High methylation levels of CpG regions were observed in the fetal placenta.

[0105]

[0106] The above 67 genes (or fragments thereof) were selected as hypermethylation biomarkers, and in the above biomarkers, the methylation level of the CpG site 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.

[0107] 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).

[0108]

[0109] 3.2. Selection of hypomethylated biomarkers for the diagnosis of Edwards syndrome

[0110] Genes or fragments thereof that have lower methylation levels in normal fetal placentas than in maternal blood and lower methylation levels in Edwards syndrome fetal placentas than in normal fetal blood were selected as hypomethylation biomarkers. Specifically, the selected hypomethylation biomarkers show a high methylation level of 0.8 or higher in maternal blood, a low hypomethylation level of △ - 0.4 to - 0.5 in normal fetuses compared to maternal blood, and a low hypomethylation level of △ - 0.2 to - 0.4 in Edwards syndrome fetuses compared to normal fetuses.

[0111] The 91 selected hypomethylated biomarkers are shown in Table 5 below.

[0112]

[0113]

[0114]

[0115] 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.

[0116] Figures 3 and 4 show the results of a heat map analysis of the levels of Edwards syndrome-specific hypomethylated biomarkers selected for Edwards syndrome diagnosis.

[0117] In the above figure, NB represents maternal blood, NT represents the placenta of a normal fetus, and T18T represents the placenta of a fetus with Edwards syndrome.

[0118]

[0119] Gene methylation levelNormal maternal bloodNormal fetal placentaEdwards syndrome fetal placentaLCK0.910.530.22FCRL50.790.580.28RHOU / RAB4A0.850.450.18GREM2 / RGS70.920.690.35DCDC2C / LINC013040.790.620.41RNU6-81P0.970.570.30CCDC1080.900.530.30LOC150935 / MIR47860.810.450.24AQP12B0.850.440.25AQP12A0.890.460.24LINC012370.900.540.35XCR10.860. 540.33FGFR30.940.620.41NDST4 / MIR19730.920.480.26TPPP0.880.570.38HNRNPH1 / C5orf600.900.520.33RASGEF1C0.910.620.35LINC01012 / LOC1001312890.920.650.44HLA-DPA10.680.280.02HLA-DPA10.750.630.03HLA-DPA10.930.650.41HLA-DPA10.780.540.18SMOC2 / THBS20.930 .590.38ADAP10.980.490.33ELFN10.850.630.36MAD1L10.900.520.31LYPD20.920.630.36MAPK150.840.520.33PUF600.880.490.19LINGO2 / LI NC012420.930.620.40APBA10.870.640.40NCS10.870.490.30FIBCD10.890.650.41FIBCD10.910.600.38FIBCD10.880.600.39RXRA / COL5A10.91 0.590.31KCNT10.800.610.37GPSM10.960.560.33PFKFB30.930.550.39ATRNL10.860.630.41FRG2B / DUX4L30.740.440.14PKP30.870.310.14MUC 5B0.900.510.38LMO10.910.570.37LOC4416010.710.270.10APOA50.910.510.39TRIM290.830.490.31B4GALNT30.940.510.36CACNA2D4LRTM20.920.570.39TSPAN9 / PRMT80.760.470.27VAMP1 / MRPL510.950.660.48GNB30.900.580.39LRIG3 / SLC16A70.900.500.30CABP10.920.520.35TMEM132D0.830.380.20LINC012570.850.550.35LOC101928416 / FBRSL10.870.490.34LINC005720.930.430.28MCF2L0.870.590.37MCF2L0.940.620.47GRTP1 / ADPRHL10.890.510.29EAPP / SNX60.910.560.35ELK2AP / KIAA01250.800.540.36ADAM6 / LINC002260.900.460.17LINC00226 / LINC002210.840.540.36C15orf480.870.600.33SV2B0.910.700.50LOC101927332 / FAM169B0.850.500.32SBK10.980.580.40RRN3P2 / SNX29P20.910.350.17ARHGEF150.910.460.25NFE2L10.960.540.33SEC14L1 / SEPT90.870.390.19B3GNTL1 / METRNL0.930.310.14AQP4-AS10.770.470.32TSHZ10.890.530.36GALR1 / LINC010290.910.570.39LINC01029, SALL30.860.570.36GRIN3B0.810.390.19SMIM24 / DOHH0.840.560.34C19orf670.950.450.29TMC40.900.550.37LILRB30.930.660.29SIRPG / LOC1002894730.940.430.27FOXS10.980.360.11HELZ20.950.570.37LOC101927797 / LINC003200.930.430.22LINC001110.920.580.42TFF20.850.470.27ZDHHC8P10.890.550.38LOC2849300.910.560.34.

[0120]

[0121] 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 lower methylation levels of CpG regions were observed in Edwards syndrome fetal placentas than in normal fetal placentas.

[0122] 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.

[0123] 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

1. A composition for diagnosing Edward syndrome, comprising a preparation for measuring the methylation level of a CpG region of a gene having a CpG region, isolated from the placenta.

2. A composition for diagnosing Edward syndrome, wherein the gene comprises at least one gene selected from the group consisting of (a) and (b) below: (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 LOC284930Name 영역을 갖는 단편.

3. In the first paragraph, 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, a composition for diagnosing Edwards syndrome.

4. A kit for diagnosing Edwards syndrome, comprising a composition according to any one of claims 1 to 3.

5. A method for providing information for diagnosing Edwards syndrome, comprising: a step of measuring the 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.

6. In claim 5, the step of measuring the methylation level is performed by at least one selected from the group consisting of PCR, methylation-specific PCR (methylation-specific polymerase chain reaction), real-time methylation-specific PCR (real-time methylation-specific polymerase chain reaction), 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 spectroscopy, and immunoprecipitation using a methylated CpG binding domain or an anti-methylcytosine antibody.

7. The 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 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, LOC284930more 영역을 갖는 단편.

8. 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 A method further comprising a step of determining Edwards syndrome when the methylation level of the gene of the above (b) is reduced compared to the methylation level of the CpG region of the same gene in maternal blood and normal placenta.

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