WO2021159722A1 - 一种预测胎盘源性疾病的评估和预测的方法和试剂盒 - Google Patents

一种预测胎盘源性疾病的评估和预测的方法和试剂盒 Download PDF

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WO2021159722A1
WO2021159722A1 PCT/CN2020/120654 CN2020120654W WO2021159722A1 WO 2021159722 A1 WO2021159722 A1 WO 2021159722A1 CN 2020120654 W CN2020120654 W CN 2020120654W WO 2021159722 A1 WO2021159722 A1 WO 2021159722A1
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placental
methylation
pregnancy
dna
dna methylation
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French (fr)
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张翼
吴凯
常璐媛
刘佳
金婉
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北京优乐复生科技有限责任公司
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Priority to EP20918272.4A priority Critical patent/EP4108779A4/en
Priority to US17/798,427 priority patent/US20230160011A1/en
Publication of WO2021159722A1 publication Critical patent/WO2021159722A1/zh

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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
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/30—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for calculating health indices; for individual health risk assessment
    • 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

  • the invention relates to the field of biological detection.
  • the present invention relates to a risk assessment method and kit for predicting pregnancy complications related to placental dysplasia. More specifically, it relates to methods and kits for assessing and predicting the risk of pregnancy-induced hypertension syndrome and other types of placental-derived diseases during the perinatal period of pregnant women in the first trimester (12-16 weeks of gestation). More specifically, the present invention relates to methods and kits for evaluating and predicting the risk of preeclampsia.
  • placenta The formation of the placenta is a vital guarantee for the normal pregnancy and healthy growth of the fetus in mammals.
  • the placenta is the interface between the mother and the fetus, which plays an important role in providing necessary nutritional support for the fetus and regulating the immune tolerance of the mother.
  • the placenta originates from the extraembryonic ectoderm (ExE). Its formation process begins at the blastocyst implantation period 6-7 days after fertilization and its development process runs through the entire pregnancy.
  • the blastocyst ready for implantation has differentiated into the inner cell mass (ICM) with the potential to develop into an embryo and the trophoectoderm (TE) with the potential to differentiate into the placental tissue.
  • the growth and development of the placenta include biological processes such as the degradation of extracellular matrix, the invasion of trophoblast cells, the differentiation of placental lineage cells, the promotion of angiogenesis, cell migration and the regulation of tolerance of the maternal immune system.
  • the development of the placenta is also accompanied by dynamic epigenetic processes, such as the remodeling process of whole-genome methylation and the regulation of expression levels by gene imprinting, which determines the fate of cell lineages and regulates genes related to placental development.
  • the expression and embryonic development play an important role.
  • DNA methylation modification is to transfer the methyl group to the fifth carbon atom of cytosine under the action of methyltransferase to form 5-methylcytosine.
  • the vast majority of DNA methylation modifications occur at CpG sites, and a very few occur at CHG and CHH sites (H stands for C, T or A).
  • Cells derived from the same developmental lineage have roughly similar DNA methylation modifications; DNA methylation modifications will change when cells undergo processes such as differentiation, transdifferentiation, and cancer; under different physiological and pathological conditions, the same cell’s DNA methylation modification may also change. Therefore, the DNA methylation modification mode can be used to calculate the source of the tissue and evaluate the physiological and pathological state of the tissue cells in which it is located.
  • DNA methylation modification is a highly dynamic process. A few hours after the formation of the fertilized egg, the erasure of DNA methylation gradually occurred in the whole genome, which reduced the average methylation level of the whole genome from about 41% after fertilization to 9% in the two-cell stage; before implantation , The genome of the blastocyst is almost completely hypomethylated. The methylation modification that has not been erased is called methylation imprinting. After blastocyst implantation, the process of DNA neo-methylation (de novo methylation) occurs, which is closely related to the regulation of the expression of genes related to embryonic development.
  • the endo-embryonic and extra-embryonic tissues show different patterns.
  • 80% of the methylation sites are differentiated in the intra-embryonic and extra-embryonic tissues.
  • Modification; for a single methylation site the DNA methylation modification of the embryonic tissue presents an "all-or-nothing" pattern, that is, the state of all methylation or all hypomethylation at a certain methylation site, However, most of the CpG sites in extraembryonic tissue cells are in a state of hemimethylation.
  • ICR can still maintain the methylation state during the eradication of whole genome methylation in the early stage of fertilized egg development. Methylated ICR can affect surrounding genes and make them also be methylated. In egg cells, the vast majority of ICRs are in a methylated state, and their distribution is mostly located on methylated islands (CpG islands, CGI) with a high density of methylated sites. In the early stages of embryonic development, gene transcription uses alternative promoters, which are located upstream of the classic promoters on CGI, and most of them are retrotransposons with long terminal repeats (LTR) , Also known as endogenous retrovirus (endoretrovirus, ERV). ERV accounts for about 8% of the human genome.
  • LTR long terminal repeats
  • Human ERV loses retrotransposon activity but retains its function as a variable promoter.
  • the transcription of CYP19A1 gene in the placenta is driven by a tissue-specific promoter-MER21 LTR, which belongs to the ERV family, allowing placental cells to express tissue-specific transcripts through alternative splicing mechanisms .
  • Preeclampsia is a common pregnancy complication that can seriously threaten the life of mothers and babies in the perinatal period. In China, the incidence of preeclampsia is roughly 2%-6%, which is the second leading cause of death among pregnant women in my country. Clinically, the typical features of preeclampsia are hypertension and proteinuria for the first time after 20 weeks of gestation, often accompanied by edema and hyperuricemia. The onset of preeclampsia can cause fatal damage to pregnant women’s liver, kidney and blood coagulation functions. Pregnant women with severe preeclampsia may develop microvascular hemolytic anemia, elevated lactate dehydrogenase and liver enzymes, headaches or other neurological symptoms.
  • preeclampsia the main reasons for the onset of preeclampsia are generally recognized as insufficient trophoblast invasion of the decidua and insufficient remodeling of the distal end of the uterine spiral artery.
  • preeclampsia the only radical cure is induction of labor.
  • a number of clinical trials have proved that taking low-dose aspirin before 16 weeks of gestation can reduce the incidence of preeclampsia.
  • In order to relieve the pressure of clinical management of preeclampsia strengthen the early prevention of preeclampsia, and reduce its incidence, it is necessary to strengthen the early screening of preeclampsia.
  • Free DNA is a single-stranded or double-stranded DNA fragment that exists in human circulating blood, urine and other body fluids. Most of its length is less than 200bp, and it exists in a very low concentration in plasma (about 1-100ng/mL).
  • cfDNA is mainly derived from apoptosis, because its fragmentation pattern is very similar to the DNA fragments produced by apoptosis. More than 90% of cfDNA comes from cells of the hematopoietic lineage.
  • the cfDNA in pregnant women's plasma is a mixture of cfDNA from the pregnant woman's own source and placenta.
  • cfDNA from fetus/placenta accounts for a small proportion (fetal source ratio: 3%-15%).
  • Existing prenatal diagnosis methods have achieved early screening of fetal genetic diseases, such as Down syndrome (T21) and other chromosomal polyploid diseases, through deep sequencing of cfDNA in pregnant women's plasma.
  • the present disclosure provides a method for detecting the methylation modification level of at least one DNA methylation modification difference site or at least one DNA methylation in a sample to be tested.
  • the present disclosure provides a kit for detecting the methylation modification level of at least one DNA methylation modification difference site or at least one DNA methylation haplotype in a sample.
  • the present disclosure provides a polynucleotide that can be used to detect the presence of DNA methylation modification.
  • the present disclosure provides a method for detecting the methylation modification level of at least one DNA methylation modification difference site or at least one DNA methylation haplotype in a test sample.
  • the present disclosure provides a method of detecting, monitoring and/or predicting whether a subject has a placental pregnancy disorder.
  • the present disclosure provides a device and a non-volatile computer-readable storage medium for detecting, monitoring, and/or predicting whether a subject has a placental pregnancy disorder.
  • the present disclosure provides a system for detecting the methylation modification level of at least one DNA methylation modification difference site or at least one DNA methylation haplotype in a test sample.
  • the present disclosure provides a system for detecting, monitoring and/or predicting whether a subject has a placental pregnancy disorder.
  • the present disclosure provides the following technical solutions.
  • a reagent for detecting at least one specific human genome region in a sample to be tested, carrying a specific DNA methylation modification or a specific DNA methylation haplotype is prepared for detection, monitoring or prediction Whether the subject has a placental-derived pregnancy disorder or the use in the kit; wherein the specific DNA methylation modification or the specific DNA methylation haplotype is derived from the placenta of normal pregnant women and Differential regions of placenta methylation in pregnant women with placental-derived pregnancy diseases.
  • the differentially methylated region is a genomic region as shown in Table 1;
  • the specific DNA methylation modification is the methylation modification of a specific CpG site in the genomic region as shown in Table 1;
  • the specific methylation haplotype is a DNA methylation haplotype covered by the genomic region as shown in Table 1;
  • the specific DNA methylation modification or specific methylation haplotype can distinguish the placenta of pregnant women with normal pregnancy and those with placental gestational disorders.
  • the detected specific DNA methylation modification or specific DNA methylation haplotype is located in one or more regions selected from the following regions (i)-(iii):
  • the LTR12 transposon is an LTR12C transposon or an LTR12E transposon;
  • the FLT1 gene and its regulatory region are the region between human chromosome chr13: 28800000bp-302000000bp;
  • LIFR gene and its regulatory region Located in the LIFR gene and its regulatory region; preferably, the LIFR gene and its regulatory region are the region between human chromosome chr5: 38370000bp-38840000bp.
  • sample to be tested is selected from blood, urine, feces, saliva, buccal swabs, cervical secretions, cervical smears
  • a sample of amniocentesis, fetal villi or fetal circulating cells preferably, the blood is peripheral blood, and more preferably, the peripheral blood is plasma.
  • the placental-derived pregnancy disorder is selected from the group consisting of gestational diabetes, twin-fetal transfusion syndrome, fetal growth restriction, gestational hypertension, and One or more of preeclampsia, severe preeclampsia, secondary preeclampsia, atypical preeclampsia, and HELLP syndrome; preferably, the pregnancy disorder is selected from the group consisting of preeclampsia, severe preeclampsia, One or more of secondary preeclampsia and atypical preeclampsia.
  • reagent is selected from DNA methylation status indicator; or
  • the reagent for detecting the presence of DNA methylation modification in the sample from the subject is a reagent required for enriching characteristic DNA.
  • the DNA methylation status indicator is selected from the group consisting of antibodies or binding proteins that recognize methylated DNA, bisulfites, enzymes with DNA catalytic oxidation, DNA deamination enzymes or methylation sensitive enzymes, or a combination thereof; preferably, the methylation sensitive enzyme is selected from methylation sensitive restriction enzymes, more preferably, the methyl group
  • the chemosensitive restriction endonuclease is selected from HpaII or BstUI, or a combination thereof.
  • reagents required to enrich the characteristic DNA are selected from the group consisting of reagents required for the hybridization probe capture method, reagents required for the polymerase chain reaction amplification method, and anchors. Determine the reagents required by the nucleic acid amplification method, the reagents used for sequencing while synthesizing, or the reagents used for single-molecule sequencing, or a combination thereof.
  • the detection is a prenatal detection; preferably, the prenatal detection is a non-invasive prenatal detection; more preferably, the The prenatal testing is non-invasive prenatal testing in the first trimester.
  • kits for detecting at least one specific DNA methylation modification or specific DNA methylation haplotype in a test sample to detect, monitor or predict whether a subject has placental origin For sexual pregnancy disorders contains:
  • the kit may also include:
  • the specific DNA methylation modification or specific DNA methylation haplotype is derived from the placenta methylation difference region of normal pregnant women and those with placental-derived pregnancy diseases.
  • the methylation modification carried may include a methylation modification of a CpG site in a specific human genome region or a specific DNA methylation haplotype.
  • the differentially methylated region is a genomic region as shown in Table 1;
  • the specific DNA methylation modification is the methylation modification of a specific CpG site in the genomic region as shown in Table 1;
  • the specific methylation haplotype is a DNA methylation haplotype covered by the genomic region as shown in Table 1;
  • the specific DNA methylation modification or specific methylation haplotype can distinguish the placenta of pregnant women with normal pregnancy and those with placental gestational disorders.
  • the reagents required to collect characteristic DNA are probes or probe sets; more preferably, the probes or probe sets are EP-007 probes or probe sets.
  • the DNA methylation status indicator is selected from the group consisting of antibodies or binding proteins that recognize methylated DNA, bisulfite, Enzymes that catalyze DNA oxidation, enzymes that have DNA deamination, or methylation-sensitive enzymes, or a combination thereof; preferably, the methylation-sensitive enzyme is selected from methylation-sensitive restriction endonucleases, More preferably, the methylation-sensitive restriction endonuclease is selected from HpaII or BstUI, or a combination thereof.
  • the sample to be tested is selected from the group consisting of blood, urine, feces, saliva, buccal swabs, cervical secretions, cervical smears Tablets, amniocentesis, a sample of fetal villi or fetal circulating cells; preferably, the blood is peripheral blood, and more preferably, the peripheral blood is plasma.
  • test is a prenatal test; preferably, the prenatal test is a non-invasive prenatal test; more preferably, the test Prenatal testing is a non-invasive prenatal testing in the first trimester.
  • a polynucleotide which may:
  • a method for detecting whether there is a DNA fragment characteristic of a placental-derived pregnancy disorder on at least one specific region of the human genome in a sample to be tested comprising a detection step, the detection step for detecting:
  • the methylation modification of the specific CpG site characteristic of the placental pregnancy disorder or the DNA methylation haplotype is derived from the placenta of a pregnant woman with normal pregnancy and the placenta of a pregnant woman with a placental pregnancy disorder. Base the difference area.
  • the DNA methylation haplotype is the DNA methylation haplotype covered by the genomic region shown in Table 1.
  • the LTR12 transposon is an LTR12C transposon or an LTR12E transposon;
  • the FLT1 gene and its regulatory region are the region between human chromosome chr13: 28800000bp-302000000bp;
  • LIFR gene and its regulatory region Located in the LIFR gene and its regulatory region; preferably, the LIFR gene and its regulatory region are the region between human chromosome chr5: 38370000bp-38840000bp.
  • a method for detecting, monitoring or predicting whether a subject has a placental-derived pregnancy disorder comprising the following steps:
  • test results of the test sample and the placental sample without the placental-derived pregnancy disorder have low similarity or the similarity statistic value is significantly lower than expected, Or the test results of the test sample and the placental sample with the placental pregnancy disorder have a higher similarity or the similarity statistic value is significantly higher than expected, then the subject has a placental pregnancy disease;
  • test results of the test sample and the placental sample without the placental-derived pregnancy disorder have high similarity or the similarity statistic value is significantly higher than expected, Or the test results of the test sample and the placental sample with the placental pregnancy disorder have low similarity or the similarity statistic value is significantly lower than expected, then the subject does not have a placental pregnancy Stage illness.
  • the sample to be tested is derived from the subject.
  • the LTR12 transposon is an LTR12C transposon or an LTR12E transposon;
  • the FLT1 gene and its regulatory region are the region between human chromosome chr13: 28800000bp-302000000bp;
  • LIFR gene and its regulatory region Located in the LIFR gene and its regulatory region; preferably, the LIFR gene and its regulatory region are the region between human chromosome chr5: 38370000bp-38840000bp.
  • the placental-derived pregnancy disorder is selected from the group consisting of gestational diabetes, twin-fetal transfusion syndrome, fetal growth restriction, and gestational hypertension , Preeclampsia, severe preeclampsia, secondary preeclampsia, atypical preeclampsia, HELLP syndrome; preferably, the pregnancy disorder is selected from preeclampsia, severe eclampsia One or more of pre-eclampsia, secondary pre-eclampsia, and atypical pre-eclampsia.
  • step (3) The method according to any one of (29) to (33), wherein the similarity statistic judgment method used in step (3) is selected from: correlation analysis (correlation), t test, Z test, Hypergeometric test, Fourier analysis, Wavelet analysis, Principal Component Analysis (PCA), Manifold Embedding (tSNE), Orthogonal Matrix Factorization (NMF), Support Vector Machine (SVM), K -Nearest Neighbor (KNN), K-means (k-means), Linear Regression (LM), Generalized Linear Regression (GLM), Gaussian Mixture Model (GMM), Neural Network (NN), Random Forest (RF), Auto-encoding Network (Autoencoder), Deep Neural Network (DNN) and their derivative variants.
  • correlation analysis correlation
  • t test Manifold Embedding
  • NMF Orthogonal Matrix Factorization
  • SVM Support Vector Machine
  • KNN K -Nearest Neighbor
  • KNN K-means (k-means)
  • the reagents required to enrich the characteristic DNA are probes or probe sets; more preferably, the probes or probe sets are EP-007 probes or probe sets.
  • the method further includes the step of extracting DNA from a sample to be tested, the sample to be tested is a peripheral blood sample; preferably, the The peripheral blood sample is a plasma sample.
  • a device for detecting, monitoring and/or predicting whether a subject has a placental pregnancy disorder including:
  • Memory for storing processors and executing instructions
  • the processor is configured to implement the method described in any one of (29)-(38) when executing the processor-executable instructions.
  • a system for detecting at least one specific methylation modification of a specific CpG site or at least one DNA methylation haplotype in a sample to be tested including a detection module, the detection module for detecting :
  • the methylation modification of the specific CpG site characteristic of the placental pregnancy disorder or the DNA methylation haplotype is derived from the placenta of a pregnant woman with normal pregnancy and the placenta of a pregnant woman with a placental pregnancy disorder. Base the difference area.
  • the DNA methylation haplotype is the DNA methylation haplotype covered by the genomic region shown in Table 1.
  • the LTR12 transposon is an LTR12C transposon or an LTR12E transposon;
  • the FLT1 gene and its regulatory region are the region between human chromosome chr13: 28800000bp-302000000bp;
  • LIFR gene and its regulatory region Located in the LIFR gene and its regulatory region; preferably, the LIFR gene and its regulatory region are the region between human chromosome chr5: 38370000bp-38840000bp.
  • a system for detecting, monitoring and/or predicting whether a subject has a placental-derived pregnancy disorder including the following modules:
  • Detection module said detection module detects:
  • Comparison module the comparison module compares:
  • test results of the test sample and the placental sample without the placental-derived pregnancy disorder have low similarity or the similarity statistic value is significantly lower than expected, Or the test results of the test sample and the placental sample with the placental pregnancy disorder have a higher similarity or the similarity statistic value is significantly higher than expected, then the subject has a placental pregnancy disease;
  • test results of the test sample and the placental sample without the placental-derived pregnancy disorder have high similarity or the similarity statistic value is significantly higher than expected, Or the test results of the test sample and the placental sample with the placental pregnancy disorder have low similarity or the similarity statistic value is significantly lower than expected, then the subject does not have a placental pregnancy Stage illness.
  • the DNA methylation haplotype is the DNA methylation haplotype covered by the genomic region shown in Table 1.
  • the LTR12 transposon is an LTR12C transposon or an LTR12E transposon;
  • the FLT1 gene and its regulatory region are the region between human chromosome chr13: 28800000bp-302000000bp;
  • LIFR gene and its regulatory region Located in the LIFR gene and its regulatory region; preferably, the LIFR gene and its regulatory region are the region between human chromosome chr5: 38370000bp-38840000bp.
  • the technical solution of the present disclosure provides a method, kit and corresponding method for detecting the methylation modification level of at least one DNA methylation modification difference site or at least one DNA methylation haplotype in a sample to be tested
  • a detection system which can be used to accurately screen in vitro whether a subject has a placental-derived pregnancy disorder.
  • Figure 1 shows a 0.8% agarose gel electrophoresis image of placental tissue gDNA.
  • Figure 2 shows the size distribution of cfDNA fragments in plasma.
  • Figure 3 shows the size distribution of Tequila V3 DNA methylation library product fragment size distribution.
  • Figure 4 shows the size of library fragments captured by the SeqCap EpiGiant probe.
  • Figure 5 shows the differentially methylated regions of the placenta of preeclampsia and the placenta of normal pregnancy.
  • Figure 6 shows the distribution map of preeclampsia-specific differentially methylated regions on the whole genome.
  • Figure 7 shows that the LTR12C transposon enriches preeclampsia-specific hypermethylated regions.
  • Figure 8 shows a schematic diagram of the changes in the methylation level of LTR12C in the human sperm-specific hypomethylated region accompanied by changes in the degree of chromatin opening; among them, the example chromatin region is chr1:212,028,092-212,163,353 (GRCh37 version) .
  • Figure 9 shows that chromatin accessibility near the hypermethylated region specific to preeclampsia increases specifically at the 8-cell stage.
  • Figure 10 shows the neomethylation defect in the placenta of preeclampsia.
  • Figure 11 shows the neomethylation defect in the LIFR gene in the placenta of preeclampsia.
  • Figure 12 shows the neomethylation defect in the FLT1 gene regulatory region in the placenta of preeclampsia.
  • Figure 13 shows that chromatin accessibility near hypomethylated areas specific to preeclampsia increases in blastocysts and placenta.
  • Figure 14 shows that the expression levels of genes near hypomethylation specific for preeclampsia are significantly increased in extravillous trophoblast cells.
  • Figure 15 shows that the DNA methylation modification level of the specific methylation region of preeclampsia can distinguish embryos (or tissues) at different developmental stages and different pregnancy complications.
  • Figure 16 shows pre-eclampsia-specific methylation haplotypes.
  • Figure 17 shows the design principle and verification of the EP-007 probe or probe set.
  • Figure 18 shows the comparison results of the target rate and the average sequencing depth of the sequencing results of EP-007 probes or probe sets captured under different combinations of hybridization and elution temperatures.
  • Figure 19 shows the effect of different hybridization times on the on-target rate of sequencing results.
  • Figure 20 shows the size of library fragments captured by EP-007 probes or probe sets.
  • Figure 21 shows the results of training a model for predicting the risk of preeclampsia.
  • Figure 22 shows the results of the sensitivity and specificity of the preeclampsia risk prediction model in the test set.
  • Figure 23 shows that the preeclampsia risk prediction model can distinguish preeclampsia subtypes of different severity.
  • Figure 24 shows that the preeclampsia risk prediction model can predict the highest systolic blood pressure in the first stage of labor during delivery.
  • the term "about” means: a value includes the standard deviation of the error of the device or method used to determine the value.
  • gestational age is a measure of pregnancy age, where the starting point is the woman's last normal menstrual period (LMP) or the corresponding age estimated by other methods.
  • LMP menstrual period
  • gestational age refers to the age of the embryo or fetus plus two weeks. In humans, although it is common for childbirth to occur within 37 to 42 weeks, childbirth usually occurs at approximately 40 weeks of gestational age.
  • infant pregnancy refers to gestational age at or before 16 weeks.
  • normal pregnancy refers to the absence of any obvious pregnancy complications during pregnancy, including but not limited to gestational hypertension, gestational diabetes, twin-fetal transfusion syndrome, fetal intrauterine growth restriction, HELLP syndrome, preeclampsia, severe preeclampsia, secondary preeclampsia, atypical preeclampsia, eclampsia.
  • control pregnancy refers to a pregnancy without pre-eclampsia, without severe pre-eclampsia, without secondary pre-eclampsia, without atypical pre-eclampsia, and without eclampsia. That is to say, the "control pregnancy” in the present disclosure may include a "normal pregnancy”.
  • placental-derived pregnancy disorder refers to a disorder during pregnancy caused by defective placental tissue development or loss of function.
  • the “placental-derived pregnancy disorder” in the present disclosure is selected from the group consisting of gestational diabetes, twin-fetal transfusion syndrome, fetal intrauterine growth restriction, gestational hypertension, eclampsia, preeclampsia, severe eclampsia Pre-eclampsia, secondary pre-eclampsia, atypical pre-eclampsia or HELLP syndrome.
  • gestational diabetes refers to the symptoms of hyperglycemia in women without diabetes before pregnancy.
  • twin blood transfusion syndrome is the most common complication in monochorionic double amniotic sac twins. Because monochorionic double amniotic sac twins share one placenta, there is a large number of vascular anastomoses in the placenta.
  • the blood of the two fetuses communicate directly.
  • One fetus is a blood supply fetus, with developmental delay, anemia, oliguria, oligohydramnios, and hypotension; the other fetus is a fetus with blood, hyperemia, urination, polyhydramnios, and cardiac hypertrophy.
  • High blood pressure, edema A blood-supplying fetus may die in utero due to fetal developmental distress; a blood-supplied fetus may also die due to heart failure.
  • fetal growth restriction means that the weight of the fetus is lower than the tenth percentile of its average weight at gestational age or two standard deviations below its average weight.
  • hypotension during pregnancy refers to the occurrence of diastolic blood pressure ⁇ 90mm Hg and/or systolic blood pressure ⁇ 140mm Hg (measured between 6 hours to 1 week) and no proteinuria and no nerves after 20 weeks of gestation.
  • preeclampsia refers to the first appearance of systolic blood pressure ⁇ 140mmHg and/or diastolic blood pressure ⁇ 90mmHg, urine protein ⁇ 300mg/24h or qualitative experiment (+) after 20 weeks of pregnancy.
  • preeclampsia may include severe preeclampsia, secondary preeclampsia, and atypical preeclampsia.
  • “severe preeclampsia” refers to the occurrence of systolic blood pressure ⁇ 160mm Hg and/or diastolic blood pressure ⁇ 110mm Hg after 20 weeks of pregnancy, and the 24-hour urine protein content is greater than or equal to 2.0g and/or qualitative experiments (+ +) Above, at least one of the following occurs at the same time: blood creatinine> 106 ⁇ mol/L or higher, platelets ⁇ 100,000/mm 3 or microvascular hemolytic anemia, elevated lactate dehydrogenase or liver enzymes, and headache or Other neurological or visual symptoms, persistent epigastric discomfort or pulmonary edema.
  • second preeclampsia refers to the occurrence of systolic blood pressure ⁇ 140mm Hg and/or diastolic blood pressure ⁇ 90mm Hg before pregnancy or 20 weeks of gestational age, and preeclampsia or eclampsia occurs during pregnancy .
  • "atypical preeclampsia” refers to the appearance of symptoms of hypertension during pregnancy and one of the following: neurological symptoms, hemolysis, elevated liver enzymes, and lower platelets; but no proteinuria (abbreviated as, That is, preeclampsia without kidney damage symptoms) or proteinuria (more than two rapid test strips are positive), and one of the following: neurological symptoms, hemolysis, elevated liver enzymes, lower platelets; but no hypertension (abbreviated name That is, preeclampsia without hypertension symptoms).
  • HELLP syndrome refers to a syndrome characterized by hemolysis, elevated liver enzymes, and thrombocytopenia that occurs during pregnancy, and is a serious complication of hypertension in pregnancy.
  • a "transposon” is also called a mobile gene, a jumping gene, which is a DNA sequence that can be inserted and excised in the genome and can change its position.
  • sequence identity and “percent identity” refer to the percentage of nucleotides that are identical (ie identical) between two or more polynucleotides.
  • sequence identity between two or more polynucleotides can be determined by the following method: aligning the nucleotide sequences of the polynucleotides and comparing the positions of the aligned polynucleotides that contain the same nucleotides The number is scored and compared with the number of positions in the aligned polynucleotide that contain different nucleotides.
  • Polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or deleted nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides).
  • Sequence identity can be calculated by dividing the number of positions containing the same nucleotide by the total number of polynucleotides. For example, the percent identity can be calculated by dividing the number of positions containing the same nucleotide by the total number of nucleotides in the polynucleotide and multiplying by 100.
  • the present disclosure relates to the stringency of hybridization conditions used to define the degree of complementarity of two polynucleotides.
  • the aforementioned polynucleotide may be selected from DNA.
  • the "stringency” used in the present disclosure refers to the temperature and ionic strength conditions during hybridization and the presence or absence of certain organic solvents. The higher the stringency, the higher the degree of complementarity between the target nucleotide sequence and the labeled polynucleotide sequence.
  • Stringent conditions refer to the temperature and ionic conditions under which only nucleotide sequences with high-frequency complementary bases will hybridize.
  • hybridizes under high stringency or very high stringency conditions describes conditions for hybridization and washing. Instructions for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6.
  • high stringency hybridization conditions means that for probes with a length of at least 100 nucleotides, follow standard Southern blotting procedures at 42°C at 5X SSPE (saline sodium phosphate EDTA), Pre-hybridization and hybridization in 0.3% SDS, 200 ⁇ g/ml sheared and denatured salmon sperm DNA and 50% formamide for 12 to 24 hours. Finally, the carrier material was washed three times with 2X SSC and 0.2% SDS at 65°C for 15 minutes each time.
  • 5X SSPE saline sodium phosphate EDTA
  • very high stringency hybridization conditions means that for probes with a length of at least 100 nucleotides, follow standard Southern blotting procedures at 42°C at 5X SSPE (saline sodium phosphate EDTA) , 0.3% SDS, 200 ⁇ g/ml sheared and denatured salmon sperm DNA and 50% formamide pre-hybridization and hybridization for 12 to 24 hours. Finally, the carrier material was washed three times with 2X SSC and 0.2% SDS at 70°C for 15 minutes each time.
  • 5X SSPE saline sodium phosphate EDTA
  • the "whole-genome bisulfite sequencing method” is also called WGBS (Whole-genome bisulfite sequencing).
  • the principle is to treat the sample to be tested with bisulfite to remove the unmethylated C bases in the genome. Converted to U, after PCR amplification, it becomes T, which is distinguished from the original C base with methylation modification, and then combined with high-throughput sequencing technology, compared with the reference sequence, you can judge CpG/CHG/CHH Whether the site is methylated, it is especially suitable for drawing a single-base resolution genome-wide DNA methylation map.
  • cfDNA free DNA
  • the length of cfDNA is mostly under 200bp, and it exists in a very low concentration (about 1-100ng/mL) in plasma.
  • genomic DNA is also referred to as “gDNA”, which refers to the total amount of DNA in an organism in a haploid state.
  • a "site” corresponds to a single site, which may be a single base position or a group of related base positions, such as a CpG site.
  • DNA methylation refers to the transfer of methyl groups to specific methyl groups with S-adenosylmethionine (SAM) as the methyl donor under the catalysis of DNA methyltransferase in the organism. Process on the base. In mammals, it is mainly the methylation of the 5'C-terminus of nucleotide cytosine residues. In the human genome, a large amount of DNA methylation occurs on the cytosine in the CpG dinucleotide, C is cytosine, G is guanine, and p is the phosphate group.
  • SAM S-adenosylmethionine
  • DNA methylation can also occur in the cytosine of nucleotide sequences such as CHG and CHH, where H is adenine, cytosine, or thymine. DNA methylation can also occur on non-cytosines, such as N6-methyladenine. In addition, DNA methylation can also be in the form of 5-hydroxymethylcytosine and the like. In most cases, DNA methylation is induced by the methylation modification on the opposite DNA strand or other CpG sites near DNA bases.
  • DNA methylation can be used as a noun to refer to DNA fragments that have been methylated.
  • demethylation means that the methylation modification on the C base of the CpG site on the original DNA fragment is erased by a biochemical process.
  • the erasure process is mainly through the TET family oxidase that specifically catalyzes the oxidation of 5-methylcytosine into 5-hydroxymethylcytosine, 5-aldehyde methylcytosine, and 5-carbonylmethyl.
  • the cytosine is then catalyzed by APOBEC family enzymes to form uracil, and finally the uracil at this site is erased and replaced with the original cytosine by DNA polymerase replication correction.
  • cent methylation refers to the DNA methylation process that occurs on a segment of DNA that does not have any methylation modification. In particular, this process is not the same as the DNA methylation induced by the methylation modification on the opposite DNA strand caused by semi-reserved replication.
  • hypomethylation means that more than 50%, preferably more than 90%, and more preferably more than 95% of C bases on the CpG site on the DNA fragment are modified by methylation.
  • “hypermethylation” and “hypermethylation” have the same meaning and can be substituted for each other.
  • hypomethylation means that less than 50%, preferably less than 10%, and more preferably less than 5% of C bases on the CpG site on the DNA fragment are modified by methylation.
  • DNA methylation site refers to a single or multiple base position where DNA methylation modification may occur.
  • CpG site, CHG site or CHH site In some cases, DNA methylation sites are equivalent to CpG sites.
  • DNA differential methylation sites can also be referred to as “DNA methylation modification sites”, which refers to a single base whose degree of DNA methylation modification differs in two sets of biological samples. Location. For example, CpG site, CHG site or CHH site.
  • DNA differential methylation region can also be referred to as “DNA methylation differential region” or “DNA methylation modification differential region”, which means that the degree of DNA methylation modification is in the two groups of biological samples.
  • a genomic region composed of multiple neighboring base positions that are different. Among them, the distance between any two adjacent base positions does not exceed 20,000 nucleotides (bases).
  • DNA methylation modification can be expressed as a single-base methylation modification, it can also be expressed as the average of multiple base methylation modifications on a continuous region, or it can be expressed It is a methylated haplotype and can also be expressed as a methylated haplotype.
  • DNA methylation modification level can be described by the overall quantitative parameter ⁇ or the abundance of methylation haplotypes.
  • the overall quantitative parameter ⁇ of DNA methylation modification level can be expressed as the average degree of methylation modification of C bases, namely:
  • methylated haplotype is also called “methylated haplotype”, which refers to the methylation modification of two or more consecutive C base positions on a single DNA fragment The combination. Because DNA methylation modifications are related on the same chromosome (DNA strand), there may be multiple adjacent C base sites, and their methylation modifications are related to each other to form a certain fixed form. Therefore, the DNA methylation haplotype is a chain pattern in which DNA methylation modifications exist on the same chromosome.
  • methylated regions of placental-derived pregnancy disorders and “placental-specific differentially methylated regions (DMRs) of placental-derived pregnancy disorders” have the same meaning and can be replaced with each other.
  • DMRs differentially methylated regions
  • the "Pearson correlation coefficient" can be used to measure the degree of correlation between two random variables, which is divided by the standard deviation of the two random variables on the basis of the covariance.
  • the Pearson correlation coefficient is a value between -1 and 1. When the linear relationship between the two variables increases, the correlation coefficient tends to 1 or -1; when one variable increases, the other variable also increases, indicating They are positively correlated, and the correlation coefficient is greater than 0; if one variable increases, the other variable decreases, indicating that there is a negative correlation between them, and the correlation coefficient is less than 0; if the correlation coefficient is equal to 0, it indicates that there is no correlation between them. There is a linear correlation.
  • similarity can be used to measure the quantitative similarity of two samples or two types of samples.
  • it can be quantitatively expressed as the probability that a sample is indeed derived from a certain type of specific sample after statistics are counted through statistical principles. In other words, it is equivalent to the probability of randomly selecting a sample from a specific sample group and finding that the sample is equivalent to a given target sample.
  • high similarity or “significantly higher” means that when a certain sample comes from a specific sample of type A but not from other specific samples (for example, not from a specific sample of B), the probability exceeds 90% , Preferably, more than 95%, more preferably, more than 99%, that is, the similarity statistic value is significantly higher than expected; “lower similarity” or “significantly lower” means that when a sample comes from a specific type A
  • the probability that the sample is not from other types of specific samples is less than 10%, preferably, less than 5%, more preferably, less than 1%, that is, the similarity statistic value is significant Lower than expected.
  • the method for measuring the degree of similarity between two samples can be selected from Bayesian method, correlation analysis, binomial distribution, beta distribution, negative binomial distribution, hypergeometric distribution, normal distribution, Poisson distribution, Komolgorov-Smirnov test (KS test), Shapiro-Wilk test.
  • the method of measuring the ratio of sample similarity ie, the probability that a certain sample is more likely to come from a specific sample of type A rather than a specific sample of type B
  • likelihood ratio test ie, the probability that a certain sample is more likely to come from a specific sample of type A rather than a specific sample of type B
  • any other appropriate mathematical statistical methods can be used to perform this type of analysis.
  • EP-007 probe or probe set is composed of a series of DNA fragments with a length of about 50-70bp and labeled ends, which can capture and enrich the genome shown in Table 1 of this disclosure. area.
  • the EP-007 probe or probe set contains all CpG sites in this specific region with a modified combination of permethylation or full demethylation, that is,
  • the EP-007 probe or probe set contains both the complementary sequence when the site is unmethylated cytosine, and the site is Complementary sequence in case of methylated cytosine.
  • the length of the probe in the "EP-007 probe or probe set" in the present disclosure is 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 , 63, 64, 65, 66, 67, 68, 69 or 70bp.
  • DNA methylation status can be used to analyze the DNA methylation status, such as microarray, real-time PCR, digital real-time PCR, and mass spectrometry (Plongthongkum et al., Nature Reviews Genetics, 2014, 15( 10): 647-61; Consortium et al., Nature Biotechnology, 2016, 34(7): 726).
  • any method or reagent that can detect DNA methylation status can be used to detect, monitor and/or predict whether a subject is suffering from There are placental-derived pregnancy disorders.
  • the methylation detection method based on the principle of bisulfite conversion has become a conventional technique for detecting DNA methylation status, and it is also well known to those skilled in the art.
  • the EZ DNA Methylation-Gold Bisulfite Conversion Kit from Zymo Research Company is used as a reagent for detecting the DNA methylation status, and the sample DNA is subjected to bisulfite conversion.
  • the inventors found that it is possible to compare the placenta sample to be tested with the methylated area of the placenta of a control pregnancy by comparing the placental sample to be tested with or without a specific placental origin.
  • Placental samples from pregnant women with the disease determine the average methylation level or methylation haplotype abundance of the sample to be tested in the placental-specific differentially methylated region of the specific placental pregnancy disorder, and then use the average methylation Level or methylation haplotype abundance analyzes the similarity between the placenta sample to be tested and the placenta with or without specific placenta-derived pregnancy conditions, so as to determine whether the source of the placental sample to be tested is related to the specific placental-derived pregnancy conditions .
  • DMRs differentially methylated regions
  • the inventors found that the placental-specific differentially methylated regions (DMRs) can be compared with the average methylation level of placental-derived pregnancy disorders to distinguish between normal pregnancy placenta and placental-derived placenta.
  • DMRs differentially methylated regions
  • the placenta of a pregnant woman with a pregnancy disorder is selected from the group consisting of gestational diabetes (GDM), twin transfusion syndrome (TTTS), fetal intrauterine growth restriction, gestational hypertension (GHT), eclampsia , Preeclampsia (PE), severe preeclampsia, secondary preeclampsia, atypical preeclampsia or placenta of patients with HELLP syndrome, and then determine whether the subject has a placental pregnancy disorder.
  • GDM gestational diabetes
  • TTTS twin transfusion syndrome
  • GHT gestational hypertension
  • PE Preeclampsia
  • severe preeclampsia secondary preeclampsia
  • atypical preeclampsia or placenta of patients with HELLP syndrome atypical preeclampsia or placenta of patients with HELLP syndrome
  • the placental pregnancy disorder is preeclampsia.
  • the placenta-specific differentially methylated regions (PE-DMRs) of preeclampsia can be divided into subgroups according to their relative methylation level, that is, compared with the regional methylation level of the placenta of a control pregnancy in the same region
  • PE-hyper-DMRs that is, genomic regions with increased levels of methylation modification in the placenta of preeclampsia
  • preeclampsia-specific hypomethylated regions PE-hypo-DMRs, That is, the genomic region where the methylation modification level is reduced in the placenta of preeclampsia).
  • the inventor found that by comparing the average methylation level of PE-DMRs regions, it is possible to distinguish germ cells or embryonic tissues at different developmental stages, including but not limited to eggs, sperm, 8-cell cleavage stage, morula, blastocyst Cell clusters during the period, the fetus at 6 weeks of pregnancy, etc.
  • PE-hyper-DMRs are mostly distributed in intergenic regions and introns. Furthermore, the inventors found that a series of human endogenous retroviruses (ERV) located between genes or introns have a higher proportion of PE-hyper-DMRs.
  • ERP human endogenous retroviruses
  • ERV enriched for preeclampsia-specific hypermethylation includes LTR12C, LTR12E, LTR13A, HERVE-int, LTR12D, LTR13, and the like.
  • the ERV that is significantly enriched in PE-hyper-DMRs specific to preeclampsia is LTR12C.
  • PE-hyper-DMRs there are regions of PE-hyper-DMRs that are hypo-methylated in human (Homo sapiens) sperm cells but are in the homology region in chimpanzees (Pan troglodytes) It is in a hyper-methylation state, that is, PE-hyper-DMRs are enriched in the specific hypomethylation region of human gametes. Furthermore, the chromatin accessibility of the PE-hyper-DMRs region is specifically increased at the 8-cell stage of human embryo development, that is, PE-hyper-DMRs are enriched in the genomic functional regions that play important functions at a specific stage of embryonic development. .
  • the inventors found that in the placenta of preeclampsia, the change in the methylation status of LTR12C is strongly related to the change in the chromatin accessibility status of the surrounding genes.
  • PE-hypo-DMRs are enriched in functional gene introns, non-coding RNA introns, 5'untranslated regions, 3'untranslated regions, and downstream regions of genes.
  • PE-hypo-DMRs are significantly enriched in the neomethylation region of the placental lineage, which indicates that the placenta of preeclampsia has obvious defects in the process of neomethylation. Furthermore, the chromatin accessibility of the PE-hypo-DMRs region is specifically increased during the morula, blastula, and placenta stages of human embryo development, and the expression of nearby genes is especially outside the villi at this embryonic development stage.
  • the trophoblast cell (EVT) rises strongly during the differentiation process. That is, PE-hypo-DMRs are enriched in the functional regions of the genome that play important functions during cell differentiation and placental development.
  • the inventors discovered that in placental tissue, there are CpG sites with a strong correlation in preeclampsia-specific methylation regions.
  • the methylation modifications carried on DNA fragments derived from such genomic regions can form a characteristic combination, that is, a preeclampsia-specific methylation haplotype.
  • the inventors found that the methylation patterns of preeclampsia-specific methylation haplotypes have obvious and stable differences between preeclampsia placenta and control pregnancy placenta. At the same time, such methylated haplotypes are not present in the plasma of normal non-pregnant women.
  • the inventors found that cfDNA fragments in the plasma of pregnant women suffering from preeclampsia can detect the methylation haplotype specific to preeclampsia.
  • the inventors discovered that the difference in the abundance of such methylated haplotypes in the plasma of pregnant women actually refers to the difference in the developmental health of the placenta.
  • the difference in the abundance of methylated haplotypes in the plasma of the test pregnant woman and the placenta with placenta-derived pregnancy disorders and the placenta of the control pregnancy can be used to determine whether the test pregnant woman has a placental-derived disease.
  • the control pregnancy-specific methylation haplotype when the abundance of preeclampsia-specific methylation haplotypes in plasma is statistically significantly lower than the abundance of preeclampsia pregnant women’s plasma, the control pregnancy-specific methylation haplotype When the abundance of methylated haplotypes is statistically significantly higher than the abundance in the plasma of pregnant women with preeclampsia, it can be considered that the placenta is in good health; while the abundance of methylated haplotypes specific to preeclampsia is relatively high At times, it can be considered that placental dysplasia is more likely to occur placental-derived diseases such as preeclampsia. Therefore, the pre-eclampsia-specific methylation haplotype discovered by the inventors can be used to predict the risk of pre-eclampsia.
  • the inventor found that it is possible to compare the test sample with a placental sample with or without the placental pregnancy disorder to compare the test sample with the placental pregnancy The average methylation level of the placental-specific differentially methylated regions of the placenta; and then by comparing the test results of the test sample and the placental sample without the placental-derived pregnancy disorder, the test is judged by the similarity of the test results Whether the person suffers from placental-derived pregnancy disorders.
  • the similarity of the test results is low (that is, the similarity statistic value is significantly lower than expected), or the test results of the test sample and the placental sample with the placental-derived pregnancy disorder have a high similarity ( That is, the similarity statistic value is significantly higher than expected), then the subject has a placental pregnancy disorder; if the test sample and the placental sample without the placental pregnancy disorder have the test results The similarity is high (that is, the similarity statistic value is significantly higher than expected), or the test results of the test sample and the placental sample with the placental-derived pregnancy disorder have low similarity (that is, the similarity statistic value Significantly lower than expected), then the subject does not suffer from placental pregnancy disorders.
  • the criterion for determining whether the similarity is higher (or significantly higher) or lower (or significantly lower) is: if the probability that the sample comes from a certain group of the samples is less than 10%, preferably less than 5 %, more preferably less than 1%, the test result is considered to have low similarity; if the probability of the sample coming from a certain group of said samples is higher than 90%, preferably higher than 95%, more preferably higher than 99% , It is considered that the similarity of the test results is relatively high.
  • the inventor found that it is possible to compare the detection result of the DNA methylation haplotype or the abundance of the DNA methylation haplotype of the sample to be tested with whether it is The DNA methylation haplotype or the detection result of the DNA methylation haplotype of the placental sample with placental gestational disorders, so as to determine whether the subject has a placental gestational period disease.
  • test results of the test sample and the placental sample that do not have the placental-derived pregnancy disorder have low similarity (that is, the similarity statistic value is significantly lower than expected), or the test sample and the placenta sample If the test results of the placental sample with the placental pregnancy disorder have high similarity (that is, the similarity statistic value is significantly higher than expected), then the subject has a placental pregnancy disorder; if the The test results of the test sample and the placental sample without the placenta-derived pregnancy disorder have high similarity (that is, the similarity statistic value is significantly higher than expected), or the test sample and the placental sample have the placental origin. The test results of the placenta samples of pregnancy disorders have low similarity (that is, the similarity statistic value is significantly lower than expected), then the subject does not suffer from placental pregnancy disorders.
  • the criterion for determining whether the similarity is higher (or significantly higher) or lower (or significantly lower) is: if the probability that the sample comes from a certain group of the samples is less than 10%, preferably less than 5 %, more preferably less than 1%, the test result is considered to have low similarity; if the probability of the sample coming from a certain group of said samples is higher than 90%, preferably higher than 95%, more preferably higher than 99% , It is considered that the similarity of the test results is relatively high.
  • the inventors used the plasma of 22 pregnant women at 11-18 weeks of gestation to establish a predictive model for the risk of preeclampsia. Specifically, the cfDNA in the plasma of pregnant women is extracted, the cfDNA fragments after the sulfite treatment are captured by a specific probe, and the DNA fragments containing the specific methylated region of preeclampsia are enriched for high-throughput sequencing. By calculating the similarity between the cfDNA sample and the placenta of preeclampsia and control pregnancy.
  • the risk of preeclampsia can be predicted by calculating the Pearson correlation coefficient between a cfDNA sample and any type of placental sample.
  • the Pearson correlation coefficient can be used to describe the degree of correlation between the preeclampsia-specific methylation haplotypes in the cfDNA of the tested pregnant women and the control pregnancy placenta and preeclampsia placenta, that is, r( CTRL) and r(PE). Furthermore, by comparing r(CTRL) and r(PE), the risk of preeclampsia during the pregnancy period of the subject pregnant women was evaluated.
  • r(CTRL)>r(PE) the risk of preeclampsia in this pregnancy is low; r(CTRL) ⁇ r(PE), the pregnant woman in this pregnancy has preeclampsia
  • the detection rate of this detection and statistical method for pregnant women with preeclampsia reached a sensitivity of 97% and a specificity of 89%.
  • the correlation coefficient can also predict the risk of pregnant women suffering from pregnancy-induced hypertension, preeclampsia, severe preeclampsia and secondary preeclampsia during the current pregnancy. Further, the correlation coefficient has a good linear correlation with the maximum systolic blood pressure of the pregnant woman in the first stage of labor, and the maximum systolic blood pressure of the labor process can be predicted through the correlation coefficient.
  • the "EP-007 probe or probe set” is composed of a series of DNA fragments with a length of 50-70 bp whose 3'ends are labeled with biotin, and its sequence includes the following four types of sequences (The design principle is shown in part a of FIG. 17 of the present disclosure):
  • C is replaced with a sequence that is reverse complementary to the sequence of T;
  • the label may be selected from being labeled at the 3'end.
  • the label may include, but is not limited to, biotin label, avidin label, fluorescent label and other labeling methods, as long as the label can make the "EP-007 probe or probe" "Needle group" can be detected by the testing instrument.
  • the detection equipment is selected from equipment capable of detecting fluorescence.
  • the content involved in the present disclosure can be implemented using any suitable biological material, and the examples in the text are only examples. It is not difficult for professionals in the field to use other biological materials or a combination of several of them, including but not limited to: blood, urine, feces, saliva, buccal swabs, cervical secretions, cervical smears, amniocentesis samples, fetuses Materials such as villus samples, fetal circulating cells, etc., realize the detection involved in the present disclosure.
  • the experimental content involved in the present disclosure can be implemented using any suitable DNA methylation modification determination method, and the examples in the text are only examples. It is not difficult for professionals in the field to use other methods or a combination of several of them, including but not limited to: sulfite conversion, enzymatic methylation modification conversion, Sanger sequencing, quantitative PCR, digital PCR, time-of-flight mass spectrometry, A DNA-specific PCR reaction, methylation-specific restriction digestion, degenerate methylation sequencing, whole-genome methylation sequencing, hybrid capture sequencing, amplicon sequencing, inverse PCR (Inverse PCR), spot imprinting, biology Methods such as chip hybridization, single-molecule sequencing, nanopore sequencing, rolling circle amplification amplification (LAMP), CRISPR enzyme-linked amplification reaction, antibody cross-linking enzyme-linked amplification reaction, etc., realize the detection involved in the present disclosure.
  • sulfite conversion enzymatic methylation modification conversion
  • Sanger sequencing quantitative PCR
  • the post-detection analysis involved in the present disclosure can be implemented using any appropriate mathematical analysis method, and the embodiments in the text are only examples. It is not difficult for professionals in the field to use other methods or a combination of several of them, including but not limited to: t-test, Z-test, hypergeometric test, Fourier analysis, Wavelet analysis, Principal component analysis (PCA), manifold embedding (tSNE), orthogonal matrix factorization (NMF), support vector machine (SVM), K-nearest neighbor (KNN), K-means (k-means), linear regression (LM ), Generalized Linear Regression (GLM), Gaussian Mixture Model (GMM), Neural Network (NN), Random Forest (RF), Autoencoder, Deep Neural Network (DNN) and its derivative variants, etc., to achieve Analysis involved in this disclosure.
  • PCA Principal component analysis
  • tSNE manifold embedding
  • NMF orthogonal matrix factorization
  • SVM support vector machine
  • KNN K-nearest neighbor
  • Example 1 Using Qiagen kit to extract gDNA from placenta tissue of pregnant women
  • Placental tissue digestion Take about 25 mg of placental tissue and prepare a digestion solution (180 ⁇ L buffer ALT + 20 ⁇ L proteinase K), mix them and incubate at 65°C for 4 hours until complete digestion.
  • a digestion solution 180 ⁇ L buffer ALT + 20 ⁇ L proteinase K
  • gDNA binding transfer all the samples after incubation to the adsorption column, centrifuge, and discard the waste liquid.
  • Washing and elution Wash the adsorption membrane with buffer AW1 and buffer AW2 in sequence, and then add the eluent to extract gDNA.
  • the first lane from the left in Figure 1 is a DNA molecular marker, that is, the ⁇ DNA product (Takara) digested by Hind III.
  • the size of the uppermost band of the DNA molecular marker is 23,130 bp.
  • the second to sixth lanes from the left are gDNA extracted from different placental tissues.
  • the electrophoretic band distribution in Figure 1 has no obvious dispersion, indicating that its integrity is good.
  • Example 1 about 25 mg of placental tissue was used to extract 8640 ng of gDNA. Measured by Qubit TM dsDNA HS Assay Kit, the average concentration is about 86.4ng/ ⁇ L. The gDNA bands in the figure are concentrated at about 20kb, indicating that the fragment size is about 20kb.
  • Example 2 Using MagMAX free DNA extraction kit to extract cfDNA from pregnant women's plasma
  • NSC stored journey MiniMax TM free DNA (cfDNA) noninvasive tube (Apostle, Inc.Silicon Valley, California. ), An outer periphery of Cell-Free DNA BCT CE blood collection tube (Streck, Inc., La Vista, NE) and the like of the blood collection tube Blood (collected from Guangdong Provincial Maternity and Child Health Hospital, clinical research approval number: Yilundi [201701044]);
  • Proteinase K (Qiagen, 19133);
  • Sample preparation Put the free DNA blood collection tube containing peripheral blood into a centrifuge to separate the plasma layer by centrifugation.
  • Plasma lysis Prepare lysis reaction solution (containing 0.4mg/ml proteinase K, plasma and 1% SDS), vortex, centrifuge and incubate at 60°C for 20 minutes. After the incubation, the lysis reaction solution was cooled to room temperature.
  • Binding of free DNA to magnetic beads Prepare a binding premix at a ratio of 3:250 MyOne magnetic beads: Binding Solution, and mix it with the plasma lysate thoroughly, vortex and shake.
  • Washing of magnetic beads After placing the reaction tube on a magnetic stand for adsorption, wash the magnetic beads twice with Wash Solution and then wash them twice with 80% absolute ethanol. After washing the magnetic beads, remove the absolute ethanol, keep the reaction tube on the magnetic stand, open the lid and let it dry.
  • the total amount of 10ng cfDNA was extracted with 2mL plasma, and its concentration was 0.5ng/ ⁇ L (determined with Qubit TM dsDNA HS Assay Kit).
  • the size distribution of cfDNA fragments in the sample was determined by the Agilent 2100 bioanalyzer.
  • Figure 2 shows the cfDNA fragment size distribution measured by the Agilent 2100 bioanalyzer. Among them, the peaks at 35bp and 10,380bp are DNA molecular markers. The main peak of cfDNA appears at about 161 bp.
  • the extracted cfDNA sample is free of gDNA contamination, and the main peak of its fragment size distribution is 161bp, which is basically consistent with the theoretical value.
  • Example 3 Using Tequila V3 DNA methylation method to build a library for gDNA or cfDNA
  • End repair Use Tequila V3 DNA methylation library building kit, and prepare end repair reaction mixture 1 according to Table 1. The corresponding PCR reaction procedure: 37°C for 30 minutes, 95°C for 10 minutes, hot lid at 105°C, volume 20 ⁇ L.
  • Ligation reaction 1 Use Tequila V3 DNA methylation library building kit to prepare reaction mixture 2 on ice according to Table 2. The corresponding PCR reaction program: 37°C for 30 minutes, 95°C for 5 minutes, 10°C hold, hot lid at 105°C, volume 30 ⁇ L.
  • Linear amplification Use Tequila V3 DNA methylation library building kit, and prepare reaction mixture 3 according to Table 3.
  • the corresponding PCR reaction program 95°C, 3min, 12X [95°C 30s; 60°C 30s; 68°C 1min], 68°C 5min, 16°C hold, hot cover 105°C, volume 100 ⁇ L.
  • Magnetic bead purification 1 Use Tequila V3 DNA methylation library building kit, 138 ⁇ L TQSB reagent and 27 ⁇ L XP magnetic beads, mix well, add the linearly amplified PCR product, incubate at room temperature and then adsorb on the magnetic rack. After adsorbing to the clarification of the liquid, remove the supernatant, add a mixture of 180 ⁇ L TQSB reagent and 100 ⁇ L DEPC water to resuspend the magnetic beads. After incubation at room temperature, it was adsorbed on a magnetic rack. After adsorbing to clarification, the supernatant was removed, the magnetic beads were washed with 80% ethanol, and the eluent was added.
  • DNA denaturation The PCR program is 95°C for 10 minutes, the lid is heated to 105°C, and the volume is 10 ⁇ L. After the program runs for 5 minutes, immediately remove the 0.2 mL PCR tube containing the sample from the PCR machine, immediately insert it on ice, and place it for 2 minutes.
  • Ligation reaction 2 Use Tequila V3 DNA methylation library building kit to prepare reaction mixture 4 on ice according to Table 4. The PCR reaction program is 25°C for 15 minutes, hot lid 35°C, volume 20 ⁇ L.
  • Magnetic bead purification 2 Add 17 ⁇ L of XP magnetic beads to the reaction product in the previous section, vortex and mix them thoroughly, and incubate at room temperature for 5 minutes. After the incubation, the supernatant was discarded after the liquid was adsorbed on the magnetic stand until the liquid was clear. After washing the magnetic beads twice with 80% ethanol, the magnetic beads were dried at room temperature and eluted with DEPC water.
  • PCR amplification The PCR amplification system is shown in Table 5. The corresponding reaction program is 98°C 45s, 12X [98°C 15s, 60°C 30s, 72°C 30s], 72°C 5min, 10°C hold, hot cover 105°C , The volume is 100 ⁇ L.
  • Magnetic Bead Purification 3 Add 17 ⁇ L XP magnetic beads to the PCR product in the previous step, mix well, and then let it stand at room temperature and incubate for 5 min. Then, it is adsorbed on the magnetic rack until the liquid is clear. After aspirating the waste solution, wash the magnetic beads twice with 80% ethanol. After aspirating and discarding the waste liquid, the magnetic beads were dried at room temperature and then eluted with DPEC water to obtain a pre-library.
  • a 70.8ng/ ⁇ L pre-library (determined with Qubit TM dsDNA HS Assay Kit) can be obtained.
  • the main peak of the fragment size distribution is about 300 bp, that is, the fragment size of the gDNA pre-library is about 300 bp ( See part b in Figure 3).
  • Example 4 Enrichment of gDNA/cfDNA genome-wide methylation sites by SeqCap EpiGiant probe
  • Hybrid elution kit for non-invasive preeclampsia early screening kit (Youle Fusheng, EU-TQ-MV1);
  • Evaporate to dryness use the hybridization elution kit of the non-invasive preeclampsia early screening kit, prepare the evaporate dry system according to Table 6 in a 1.5mL EP tube and place it in a vacuum concentrator, open the lid and evaporate to dryness at 60°C. Among them, the library samples were constructed according to the method described in Example 3.
  • Hybridization reaction Use the hybridization elution kit of the non-invasive preeclampsia early screening kit, prepare the hybridization system according to Table 7 in a 200 ⁇ L PCR tube, vortex and mix, and then stand at room temperature for 10 minutes. After that, the denaturation was performed in a PCR machine at 95°C for 10 minutes. When the denaturation is 8 minutes, the PCR tube containing 4.5 ⁇ L of the probe is placed on the PCR machine at 95° C., and the probe is preheated. After the denaturation, the probe was added to the hybridization system mixture at 47°C. After mixing by vortex, place in a 47°C PCR machine (hot lid temperature is 57°C) for 72 hours for hybridization reaction.
  • Capture and cleaning Take 100 ⁇ L of streptomycin-modified magnetic beads (Dynabeads TM M-270 streptavidin) equilibrated at room temperature, 200 ⁇ L of magnetic bead washing buffer (1X TQWS5 reagent) and the reaction product of the previous step, and then mix it well. Adsorb on the magnetic stand until the liquid is clear. Aspirate the liquid, wash the magnetic beads once with 200 ⁇ L magnetic bead washing buffer, then resuspend the magnetic beads with an equal volume of magnetic bead washing buffer, and place on a magnetic stand to absorb until the liquid is clear. After aspirating and discarding the waste liquid, place it in a PCR machine and incubate for 45 min.
  • Aspirate and discard the waste liquid add 180 ⁇ L 1X TQWS3, shake and mix at room temperature for 1 min, then place it on a magnetic stand to absorb until the liquid is clear. Aspirate and discard the waste solution, add 23 ⁇ L of TQNF reagent, shake and mix well.
  • Amplification of the captured product Use the hybridization elution kit of the non-invasive preeclampsia early screening kit to prepare the capture product amplification system according to Table 8.
  • the corresponding PCR reaction program is 98°C 45s, 8X ⁇ 98°C 15s; 60 °C 30s; 72°C 30s] 72°C 2min, 16°C hold, hot cover 105°C, volume 50 ⁇ L.
  • reaction system Single sample dosage ⁇ L TQAE2 enzyme 25 TQPR3 reagent 2 Capture product (including magnetic beads) twenty three total capacity 50
  • the above reaction product is placed on a magnetic stand to absorb until the liquid is clarified, and the supernatant is sucked and mixed with 90 ⁇ L of XP magnetic beads by vortex. After standing at room temperature for 5 minutes, it is placed on a magnetic stand and absorbed until the liquid is clear. Aspirate and discard the waste liquid, wash the magnetic beads twice with 80% ethanol, and open the lid to dry at room temperature. After the magnetic beads are dry, add 25 ⁇ L of TQEB reagent and incubate at room temperature for 5 minutes, and then adsorb on the magnetic rack until the liquid is clear. Pipet the clear liquid into a new EP tube to be the purified captured product.
  • the library can be enriched by 4.46 ng/ ⁇ L after being captured by the SeqCap EpiGiant probe as determined by Qubit TM dsDNA HS Assay Kit.
  • Figure 4 shows that the Agilent 2100 bioanalyzer measures the size of the fragments of the SeqCap EpiGiant probe capture library, and the size of the aforementioned fragments is about 300 bp.
  • part a in Figure 4 shows the library constructed by placental gDNA, and its fragment size is about 300 bp.
  • Part b in Figure 4 shows the library constructed from cfDNA in plasma, and its fragment size is about 300 bp.
  • Example 5 Quality detection of data after second-generation high-throughput sequencing on the Illumina platform of the methylation library
  • the DNA prelibrary was sequenced on the computer according to the instructions of the Illumina platform kit. In the offline data of sequencing, the ratio of Q30 is greater than 80%. Split the sample using bcl2fastq (0.2.1). The quality control standard is unknown, the number of fragments separated is ⁇ 5%, and the average fragment length is >160bp.
  • the PhiX library uses Euler-PhiX process quality control, and the eligibility standard is error rate ⁇ 0.001%.
  • the sub-library uses Fastqc (3.2) for quality control.
  • Example 6 Placenta-specific differentially methylated regions of preeclampsia
  • Example 1 The procedures of placental gDNA extraction, SeqCap EpiGiant probe capture and high-throughput sequencing are the same as in Example 1, Example 4 and Example 5.
  • CpG site methylation level analysis PileOMeth (version number: 0.1.13) was used to extract the sequencing depth and methylation level of each CpG site.
  • Quality control conditions Sequencing depth ⁇ 200X on a single CpG site is required.
  • Requirements It is subject to the following conditions: the distribution of methylation modification degree in preeclampsia placenta samples is significantly different from that of control placenta samples, p ⁇ 0.001.
  • the methylation levels of CpG sites obtained through the above analysis showed significant and stable differences in methylation levels between different groups of samples, namely the placenta of preeclampsia, the placenta of control pregnancy, and the plasma group of non-pregnant women.
  • the analysis above The obtained CpG site is the specific methylation modification site of the placenta of preeclampsia, see Appendix Table 1 for details.
  • Tissue homogenate Take 20mg of frozen tissue in a pre-cooled glass homogenizer, and add 1ml of pre-cooled 1x HB buffer (as shown in Table 10, where the 1x HB buffer shown in Table 10 passes through Table 9 The 1.022xHB buffer solution was further prepared) and then the tissue was ground. The ground tissue homogenate was passed through a 70-micron cell sieve and transferred to a low-adsorption tube, and centrifuged at 4°C for 5 min. Discard the supernatant and resuspend in fresh HB buffer.
  • Iodixanol gradient centrifugation add an equal volume of 50% iodixanol to the resuspension solution, prepare 30% and 40% iodixanol and construct 25%/30%/40% iodixanol Density gradient centrifugation system for enriching cell nuclei, centrifugation at 4°C and 3000 rcf for 20 minutes. After centrifugation, the nucleus layer located at the interface of 30% and 40% iodixanol was drawn. Add RSB buffer (Table 11) to dilute the nuclei.
  • Tn5 transposase digestion for library construction and purification After centrifugation at 4°C at 500 rcf for 10 minutes, perform Tn5 transposase digestion according to the instructions of the VazymeTD501-02 TruePrepDNALibraryPrepKit kit. Purify the digested product according to Zymo D4061 Oligo Clean&Concentrator kit. Perform Index PCR on the purified product according to the TruePrep Index Kit V2 for Illumina kit. The PCR products were purified by using XP magnetic beads.
  • Use deeptools (version number 3.3.0) to extract coverage
  • use R software version number 3.5.2) to analyze transcription factors and TSS area coverage values
  • use macs2 software version number 2.2.4
  • Genrich software version number 0.6
  • diffBind and NarrowPeaks R language package from Bioconductor
  • the horizontal axis represents the average methylation level of the placenta-specific differentially methylated area of the normal pregnancy placenta
  • the vertical axis represents the average methylation level of the placenta of the preeclampsia in the same area.
  • the density distribution curve in Figure 5b describes the differentially methylated region in Figure 5a. Therefore, as shown in part b of FIG. 5, the density distribution curve of the indifference area (light gray) and the methylated area (dark gray) with significant statistical difference in part a of FIG.
  • PE-DMRs pre-eclampsia-specific differentially methylated regions
  • PE-DMRs As shown in Figure 6, comparing PE-DMRs to the whole gene, it is found that the distribution of PE-DMRs is concentrated in gene regulatory regions, such as non-coding transcribed regions (5'UTR/3'UTR), intergenic regions (intergenic regions) , Intron (intron) and part of the exon region. Further statistical enrichment analysis showed that PE-hypo-DMRs were significantly enriched in introns, 5'UTR/3'UTR and non-coding RNA intron regions; while PE-hyper-DMRs were mostly distributed in genes Between regions and intron regions, their distribution patterns are random.
  • LTR12 family transposons are significantly enriched in PE-hyper-DMRs.
  • about 30% of LTR12C transposons contain PE-hyper-DMRs.
  • Example 7 Screening of new methylation regions specific to the placental lineage
  • CpG site methylation level analysis PileOMeth (version number 0.1.13) was used to extract the sequencing depth and methylation level of each CpG site.
  • Quality control conditions A single CpG site is required to be ⁇ 200X.
  • Use R language (3.5.2) to analyze each methylation site, screen the placenta-specific hypermethylation modification site and this site is in a state of hypomethylation in the white blood cells of healthy non-pregnant women.
  • the differentially modified methylation sites selected by the foregoing analysis are combined with their regions, and the methylation levels in the entire region are averaged, and then the placental-specific hypermethylated regions are screened out.
  • HUVEC a differentiated human placental cell line
  • hESC1 an undifferentiated human embryonic stem cell line
  • DNA methylation differentially modified sites can screen out hypermethylation and hypermethylation in HUVEC.
  • the t-test was used to perform statistical tests on the differentially methylated sites, which must meet p ⁇ 0.001.
  • the selected differentially modified methylation sites are merged into regions, and the methylation levels in the entire region are averaged, and then HUVEC-specific hypermethylated regions are screened out.
  • the placental-specific hypermethylated region and the HUVEC-specific hypermethylated region are taken as the intersection of the placental lineage-specific neomethylated region.
  • Example 8 Preeclampsia-specific hypomethylated regions are enriched in the newly methylated regions of the placental lineage
  • RNA expression time series analysis Monocle (version number 0.2.0) was used to extract RNA expression data, and principal component analysis (reduce_dimension function, Monocle) and UMAP method were used to reduce the dimensionality of the data. For each cell, calculate the average expression level of genes of the same family (genes in the range of 50 kbp near the position of PE-hypo-DMR or PE-hyper-DMR) as the PE-hypo-DMR gene and PE-hyper- The expression level of the DMR gene.
  • VCT placental villous cytotrophoblasts
  • SCT syncytial trophoblasts
  • EVT extravillous trophoblasts
  • Example 9 The average methylation level of the specific methylated regions of preeclampsia can distinguish germ cells or embryos at different developmental stages Fetal tissue
  • Example 4 The experimental method and data analysis of DNA methylation sequencing on the placenta are the same as in Example 4, Example 5 and Example 6.
  • the PE-hyper-DMRs and PE-hypo-DMRs regions can be obtained through the experiment and analysis of the above examples.
  • the average methylation level in the hypermethylated area and hypomethylated area specific to preeclampsia can make the placenta and early embryo/fetus of preeclampsia perform well. distinguish.
  • Part a and part b in Figure 15 are the average methylation of embryos (or tissues) at different developmental stages and the placenta of different pregnancy complications on the hypermethylated and hypomethylated regions specific to preeclampsia, respectively level.
  • Part c in Figure 15 is the average methylation level of the hypermethylated region specific in preeclampsia as the abscissa, and the average methylation level of the hypomethylated region specific in preeclampsia as the ordinate.
  • Example 10 The average methylation level of the specific methylation area of preeclampsia can distinguish the placenta of different pregnancy complications
  • PE-hyper-DMRs and PE-hypo-DMRs areas are effective for hypertension during pregnancy, diabetes during pregnancy, twin-fetal transfusion syndrome, and those without placental gestational disorders.
  • Statistics of the placenta and the average methylation level of the placenta in the above-mentioned areas of the placenta and preeclampsia showed that the average methylation level of the specific hypermethylated area and hypomethylated area of preeclampsia can distinguish different pregnancy complications ( Figure 15c), so PE-hyper-DMRs and PE-hypo-DMRs have specific methylation modification differences in the placenta of preeclampsia.
  • Example 11 Differences in methylation haplotypes between preeclampsia group and control pregnancy group
  • Example 1 The extraction of placental tissue gDNA/plasma cfDNA and the construction process of DNA library are roughly the same as in Example 1, Example 2, Example 4, and Example 5.
  • Basic haplotype which requires that its frequency of occurrence in the placenta of preeclampsia is at least 20 times that of the control placenta; or that its frequency of occurrence in the control placenta is at least in the placenta of preeclampsia 20 times the frequency.
  • the selected methylation haplotypes are shown in Attached Table 3.
  • the preeclampsia-specific methylation haplotypes listed in the table have significant and stable differences in frequency between control pregnancy placenta and preeclampsia placenta, for example Shown in Figure 16.
  • part a and part b of Fig. 16 respectively illustrate the methylation haplotypes of hypermethylated regions and hypomethylated regions specific to preeclampsia.
  • Example 12 Design and verification of EP-007 probe or probe set
  • KAPA double-stranded DNA library building kit KAPA HTP Lib PrepKit, KK8235
  • the basic principle of the EP-007 probe or probe set design is as follows: the probe consists of several 59bp long and specific methylation haplotypes after sulfite sequencing.
  • the site sequence is composed of complementary sequences.
  • each CpG site will synthesize one methylated (G) and unmethylated (A) probe corresponding to the site.
  • the two probes covering adjacent sites contain 40 bp overlapping sequences.
  • the pre-eclampsia-specific methylation haplotypes obtained through the foregoing analysis were probed for the genomic region where they were located, and complementary DNA probes were synthesized (Twist Inc., USA). As shown in part a in Figure 17, it is basically designed as a 59 bp length DNA complementary strand.
  • the sequence of the target area to be detected is:
  • This sequence is part of the upstream region of the FLT1 gene. Based on the design principle of the probe described in this disclosure, the probe designed for the aforementioned area is:
  • the synthesized DNA probe follow the operating instructions of the KAPA double-stranded DNA library construction kit to perform double-stranded library construction and sequencing. As shown in Table 12, the proportion of data less than 1X sequencing depth is almost 0 and the standardized average sequencing depth is almost 0 if the average sequencing depth is below 0.2, so the probe can cover the selected area well.
  • the sequencing results of EP-007 probes or probe sets after double-stranded library construction are shown in part b of Figure 17, the sequencing results of EP-007 probes or probe sets after double-stranded library construction.
  • the abscissa is the ratio of the sequencing depth of different probe fragments to the average sequencing depth, and the ordinate represents the distribution density.
  • the sequencing depth distribution conforms to the Poisson distribution. Therefore, the synthesis quality of EP-007 probes or probe sets is unbiased and reliable.
  • Example 13 Optimizing the hybridization time of EP-007 probe or probe set and the reaction temperature of hybridization and elution
  • the hybridization elution kit of the non-invasive preeclampsia early screening kit (Youle Fusheng, EU-TQ-MV1);
  • Part a and part b in Figure 18 respectively show the on-target sequencing results at different hybridization temperatures (47°C, 56°C, and 65°C) and elution temperatures (47°C, 56°C, and 65°C). Rate and average sequencing depth.
  • the circle is for hybridization at 47°C
  • the square is for hybridization at 56°C
  • the triangle is for hybridization at 65°C.
  • Figure 19 shows the target rate of library products with different hybridization times (4h, 8h, 12h, 24h, 36h, 48h and 60h) at an elution temperature of 56°C during hybridization. Sequencing results with a target rate greater than 30%.
  • the hybridization time of more than 12h can obtain more than 30% on-target sequencing results.
  • Example 14 Using EP-007 probes or probe sets to capture the differential sites of gDNA or cfDNA preeclampsia methylation modification
  • the hybridization elution kit of the non-invasive preeclampsia early screening kit (Youle Fusheng, EU-TQ-MV1);
  • Figure 20 shows the fragment size distribution of the library captured by the probe as measured by the Agilent 2100 bioanalyzer. The peaks at 35bp and 10,380bp are DNA molecular markers. Wherein, part a of Fig. 20 shows the fragment size of the library constructed from placental gDNA, and part b of Fig. 15 shows the fragment size of the library constructed from cfDNA in plasma.
  • the library can be enriched at 2.98ng/ ⁇ L by EP-007 probe or probe set capture.
  • the fragment size of the library constructed by placental gDNA and the library constructed by cfDNA in plasma captured by EP-007 probe or probe set is about 300 bp.
  • Proteinase K (Qiagen, 19133);
  • linear regression analysis of r(CTRL) and r(PE) with the clinical diagnosis of 22 pregnant women can obtain linear scores, which can be used to assess the degree of placental dysplasia associated with preeclampsia, that is, suffering from eclampsia.
  • the placental dysplasia of the pregnant women in the early stage is high, correspondingly, the linear score is high; while the pregnant women of the control pregnancy have relatively good placental development, so the linear score is low.
  • FIG. 16 shows the linear score calculated by correlation analysis and linear regression statistics for the analysis of the methylation level of the PE-DMRs region.
  • Example 16 Retrospective clinical sample verification of preeclampsia risk prediction model
  • Example 13 The same as in Example 13, in which a total of 159 preeclampsia or control pregnancy pregnant women with peripheral blood samples drawn from 10-38 gestational weeks (Guangdong Maternity and Child Health Hospital Yilundi [201701044]) were used.
  • preeclampsia-specific methylation haplotype regions in Appendix 3 calculate the average methylation level of the subjects in each region to obtain the matrix M(test), and calculate M(test) and M(CTRL) respectively. ) And the Pearson correlation coefficient of M(PE), namely r(CTRL) and r(PE). Then compare r(CTRL) and r(PE) to predict the risk of preeclampsia. If r(CTRL)>r(PE), the risk of preeclampsia is high; r(CTRL) ⁇ r(PE), Low risk of preeclampsia.
  • the results show that the method can distinguish between pregnant women with pre-eclampsia and control pregnant women, with a sensitivity of 97% (36/37) and a specificity of 89% (109/122).
  • Example 17 Preeclampsia risk prediction model can distinguish preeclampsia subtypes of different severity
  • Example 13 Same as Example 13, in which a total of 159 pre-eclampsia or normal pregnant women with peripheral blood samples taken during gestational weeks 10-38 (collected from Guangdong Provincial Maternity and Child Health Hospital, clinical research approval number Yilundi [201701044])
  • Example 18 Pre-eclampsia risk prediction model can predict the highest pressure in the first stage of labor during pregnancy
  • Example 13 Same as Example 13, in which a total of 181 preeclampsia or control pregnancy pregnant women with peripheral blood samples taken during gestational weeks 10-38 (collected from Guangdong Provincial Maternity and Child Health Hospital, clinical research approval number Yilundi [201701044] )
  • Genomic region of chromosome 1 Genomic region of chromosome 1:
  • Genomic region of chromosome 10 Genomic region of chromosome 10:
  • Genomic region of chromosome 11 Genomic region of chromosome 11:
  • Genomic region of chromosome 12 Genomic region of chromosome 12:
  • Genomic region of chromosome 13 Genomic region of chromosome 13:
  • Genomic region of chromosome 15 Genomic region of chromosome 15:
  • Genomic region of chromosome 16 Genomic region of chromosome 16:
  • Genomic region of chromosome 18 Genomic region of chromosome 18:
  • Genomic region of chromosome 19 Genomic region of chromosome 19:
  • Genomic region of chromosome 2 Genomic region of chromosome 2:
  • Genomic region of chromosome 20 Genomic region of chromosome 20:
  • Genomic region of chromosome 21 Genomic region of chromosome 21:
  • Genomic region of chromosome 3 Genomic region of chromosome 3:
  • Genomic region of chromosome 5 Genomic region of chromosome 5:
  • Genomic region of chromosome 6 Genomic region of chromosome 6:
  • Genomic region of chromosome 8 Genomic region of chromosome 8:
  • Genomic region of chromosome 9 Genomic region of chromosome 9:

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Abstract

本公开涉及一种预测胎盘源性疾病的评估和预测的方法和试剂盒。具体的，本公开的技术方案提供了一种检测待测样品中给定基因组位置上至少一个DNA甲基化修饰差异位点的甲基化修饰水平或至少一种DNA甲基化单倍型的方法、试剂盒以及相应的检测系统，其可以用于准确的体外筛查受试者是否患有胎盘源性妊娠期病症。

Description

一种预测胎盘源性疾病的评估和预测的方法和试剂盒 技术领域
本发明涉及生物检测领域。具体的，本发明涉及用于预测与胎盘发育不良相关的妊娠并发症的风险评估方法和试剂盒。更具体的，其涉及在早孕周(孕12-16周)对孕妇在围产期罹患妊娠期高血压综合症及其它类型胎盘源型疾病的风险进行评估和预测的方法和试剂盒。更具体的，本发明涉及对子痫前期风险的评估和预测的方法和试剂盒。
背景技术
胎盘的形成是哺乳动物正常妊娠和胎儿健康生长至关重要的保障。胎盘是母胎界面，发挥着为胎儿提供必要的营养支持和调节母体的免疫耐受等重要作用。从胚胎发育的角度，胎盘起源于胚外外胚层(extraembryonic ectoderm,ExE)。其形成过程开始于受精后6-7天的囊胚(blastocyst)着床期而其发育过程贯穿整个妊娠期。准备着床的囊胚已分化成具有发育成胚胎潜能的内细胞团(inner cell mass,ICM)和具有分化成胎盘组织潜能的滋养外胚层(trophoectoderm,TE)。胎盘的生长发育包括细胞外基质的降解、滋养层细胞入侵、胎盘谱系细胞分化、促进血管生成、细胞迁移和调节母体免疫系统的耐受等生物学过程。除此之外，胎盘的发育也伴随着动态的表观遗传学过程，如全基因组甲基化的重塑过程和基因印记对表达水平的调控，对决定细胞谱系的命运、调控胎盘发育相关基因的表达和胚胎发育有着重要的作用。
DNA甲基化修饰是在甲基转移酶的作用下将甲基基团转移至胞嘧啶的五号位碳原子上而形成5-甲基胞嘧啶。绝大部分DNA甲基化修饰发生在CpG位点上，极少数发生在CHG、CHH位点上(H代表C，T或A)。来源于同一发育谱系的细胞，DNA甲基化修饰大致类似；在细胞发生分化、转分化、癌变等过程时，DNA甲基化修饰会发生改变；在不同的生理病理情况下，同一种细胞的DNA甲基化修饰也可能发生改变。故可通过DNA甲基化修饰的模式来推算其组织来源及评估其所在组织细胞所处的生理病理状态。
在受精卵发育过程中，DNA甲基化修饰是一个高度动态的过程。在受精卵形成几个小时后，全基因组范围内逐步发生DNA甲基化擦除使得全基因组平均甲基化的水平从受精后的约41％下降至二细胞期的9％；在着床前，囊胚的基因组几乎处于完全低甲基化的状态。未被擦除的甲基化修饰，被称为基因印记修饰(methylation imprinting)。囊胚着床后会发生DNA新生甲基化(de novo methylation)的过程，与调控胚胎发育相关基因的表达密切相关。在新生甲基化的过程中，胚内组织和胚外组织呈现出不同的模式，在全基因组水平上其表现为，80％的甲基化位点在胚内和胚外组织中被差异化地修饰；对单个甲基化位点，胚内组织的DNA甲基化修饰呈现“全或无”模式，即某一甲基化位点上全部甲基化或全部低甲基化的状态，然而胚外组织细胞的CpG位点大部分处于半甲基化状态。
对于绝大多数位于常染色体上的基因，从父源和母源继承来的等位基因被同时表达。然而，存在少数基因表达父源或母源的等位基因，这种单等位基因表达，即基因印记(genomic imprinting)的现象是通过不同亲本间不对称的表观遗传学修饰而实现的。印记基因的发生对正常的组织分化和胚胎发育有重要意义。例如，胎儿宫内生长受限(fetal growth restriction，FGR)与IGF2上的基因印记的缺失进而导致胎盘过小或绒毛发育不全有关。基因印记的过程可以通过父源和母源基因组的差异甲基化修饰而实现，被印记的基因的甲基化修饰是由印记控制区域(imprinting control region，ICR)调控。ICR可以在受精卵发育早期的全基因组甲基化擦除过程中仍然维持甲基化的状态，甲基化的ICR可影响周围基因使其同样被甲基化修饰。在卵子细胞中，绝大多数的ICR处于甲基化的状态，其分布多位于含高密度的甲基化位点的甲基化岛(CpG island，CGI)。在胚胎发育的早期，基因转录会使用可变启动子(alternative promoter)，其位于CGI上的经典启动子的上游，其多为具有长末端重复序列(long terminal repeat，LTR)逆转录转座子，也被称为内源性逆转录病毒(endoretrovirus，ERV)。ERV在人基因组中约占8％，人源ERV丢失了逆转录转座子活性但保留了其作为可变启动子的功能。例如，CYP19A1基因在胎盘中的转录是由组织特异的启动子－－MER21 LTR驱动的，该启动子属于ERV家 族，使胎盘细胞通过选择性剪切(alternative splicing)的机制表达组织特异的转录本。
子痫前期(preeclampsia，PE)是一种常见的可严重威胁围产期母婴生命的妊娠并发症。在中国，子痫前期的发病率大致为2％－6％，是导致我国孕妇死亡的第二大原因。在临床上，子痫前期的典型特征是20孕周后首次出现高血压和蛋白尿，常伴有水肿和高尿酸血症。子痫前期发病会导致孕妇肝功能、肾功能及凝血功能发生致命性的损伤，重度子痫前期孕妇或出现微血管溶血性贫血，乳酸脱氢酶、肝酶升高，头痛或其它神经系统症状。
目前普遍认同的子痫前期发病的主要原因是滋养层细胞(trophoblast)入侵子宫蛻膜不足和子宫螺旋动脉远端重塑不足。然而目前临床上仍缺乏对子痫前期的治疗手段，唯一的根治手段是引产。多项临床实验证明，在孕16周前服用低剂量的阿斯匹林可降低子痫前期的发病率。为缓解子痫前期的临床管理压力、加强对子痫前期的早期预防、降低其发病率，需加强对子痫前期的早期筛查。已有的科学研究表明，刚果红(Congo red)染料可以结合子痫前期孕妇尿液中的β样淀粉蛋白而显红色；子痫前期患者血浆中与胎盘发育相关的蛋白PlGF含量降低而sFlt1含量升高，故刚果红染色结果及PlGF/sFlt1的比值可作为临床诊断、预测子痫前期的指标。然而，以上的生物化学检测方法仅适用于25周或以上孕周人群，对于小于16孕周人群不适用。由于阿司匹林干预仅在16孕周前有效，因此以上方法对指导医生用药、预防子痫前期发生起着较为有限的作用。已有的科研成果显示，子痫前期胎盘较正常妊娠胎盘存在显著的DNA甲基化修饰差异。
游离DNA(cfDNA)是存在于人的循环血、尿液及其他体液中的的单链或双链DNA片段。其长度大多为200bp以下，在血浆中以很低的浓度存在(约1-100ng/mL)。cfDNA主要来源于细胞凋亡，因其片段化的模式与细胞凋亡所产生的DNA片段非常相似。90％以上的cfDNA来自造血系统谱系细胞。孕妇血浆中的cfDNA是孕妇自身来源和胎盘来源的cfDNA的混合物，其中胎儿/胎盘来源的cfDNA占较小比例(胎源比例：3％-15％)。现有的产前诊断方法通过对孕妇血浆中cfDNA的深度测序已实现对胎儿遗传疾病，如唐氏综合症(T21)等染色体多倍疾病的早期筛查。
在现有技术中，已开展的大量临床试验单独或联合利用生物化学、生物物理检测及流行病统计学来预测胎盘发育不良相关的妊娠并发症的风险，但受限于其灵敏度和特异性也未能实现在较早孕周对前述疾病的风险的发病进行有效评估预测，迄今尚未有成熟可靠的筛查手段应用于临床。
发明内容
发明要解决的问题
基于现有技术中存在的缺陷，在一个实施方式中，本公开提供一种检测待测样品中的至少一种DNA甲基化修饰差异位点的甲基化修饰水平或至少一种DNA甲基化单倍型的试剂在制备用于检测、监测和/或预测受试者是否患有胎盘源性妊娠期病症的试剂或试剂盒中的用途。
在另一个实施方式中，本公开提供一种试剂盒，其用于检测样品中至少一种DNA甲基化修饰差异位点的甲基化修饰水平或至少一种DNA甲基化单倍型。
在另一个实施方式中，本公开提供一种多核苷酸，其能够用于检测DNA甲基化修饰的存在。
在另一个实施方式中，本公开提供一种检测待测样品中至少一种DNA甲基化修饰差异位点的甲基化修饰水平或至少一种DNA甲基化单倍型的方法。
在另一个实施方式中，本公开提供一种检测、监测和/或预测受试者是否患有胎盘源性妊娠期病症的方法。
在另一个实施方式中，本公开提供一种用于检测、监测和/或预测受试者是否患有胎盘源性妊娠期病症的设备和非易失性计算机可读存储介质。
在另一个实施方式中，本公开提供一种检测待测样品中至少一种DNA甲基化修饰差异位点的甲基化修饰水平或至少一种DNA甲基化单倍型的系统。
在另一个实施方式中，本公开提供一种检测、监测和/或预测受试者是否患有胎盘源性妊娠期病症的系统。
用于解决问题的方案
本公开提供了如下技术方案。
(1)一种检测待测样品中的至少一个特定人类基因组区域上的、携带特定的DNA甲基化修饰或特定的DNA甲基化单倍型的试剂在制备用于检测、监测或预测受试者是否患有胎盘源性妊娠期病症的试剂或试剂盒中的用途；其中，所述特定的DNA甲基化修饰或所述特定的DNA甲基化单倍型来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域。
(2)根据(1)所述的用途，其中，所述待测样品来源于所述受试者；
所述甲基化差异区域为如附表1所示的基因组区域；
所述特定的DNA甲基化修饰为如附表1所示的基因组区域内特定CpG位点的甲基化修饰；
所述特定的甲基化单倍型为如附表1所示的基因组区域所覆盖的DNA甲基化单倍型；
所述特定的DNA甲基化修饰或特定的甲基化单倍型能区分正常妊娠孕妇胎盘和患有胎盘源性妊娠期病症的孕妇胎盘。
(3)根据(1)-(2)任一项所述的用途，其中，所述待测样品来源于所述受试者；
所检测的所述特定的DNA甲基化修饰或特定的DNA甲基化单倍型位于选自如下(i)-(iii)所示的区域中的一个或多个区域：
(i)位于LTR12转座子家族区域；优选的，所述LTR12转座子为LTR12C转座子或LTR12E转座子；
(ii)位于FLT1基因及其调控区域；优选的，所述FLT1基因及其调控区域为人类染色体chr13:28800000bp-302000000bp之间的区域；
(iii)位于LIFR基因及其调控区域；优选的，所述LIFR基因及其调控区域为人类染色体chr5:38370000bp–38840000bp之间的区域。
(4)根据(1)-(3)任一项所述的用途，其中，所述待测样品选自来源于血液、尿液、粪便、唾液、口腔拭子、宫颈分泌物、宫颈涂片、羊水穿刺、胎儿绒毛或胎儿循环细胞的样品；优选的，所述血液为外周血，更优选的，所述外周血为血浆。
(5)根据(1)-(4)任一项所述的用途，其中，胎盘源性妊娠期病症选自妊娠期糖尿病、双胎输血综合征、胎儿生长受限、妊娠期高血压、子痫前期、重度子痫前期、继发型子痫前期、非典型子痫前期、HELLP综合症中的一种或多种；优选的，所述妊娠期病症选自子痫前期、重度子痫前期、继发型子痫前期、非典型子痫前期中的一种或多种。
(6)根据(1)-(5)任一项所述的用途，其中，其中，检测所述来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域的试剂选自DNA甲基化状态显示剂；或
检测来自受试者样品中DNA甲基化修饰的存在的试剂；
优选的，所述检测来自受试者样品中DNA甲基化修饰的存在的试剂为富集特征DNA所需的试剂。
(7)根据(6)所述的用途，其中，所述DNA甲基化状态显示剂选自识别甲基化DNA的抗体或结合蛋白、亚硫酸氢盐、具有DNA催化氧化作用的酶类、具有DNA脱氨基作用的酶类或甲基化敏感酶，或其组合；优选的，所述甲基化敏感酶选自甲基化敏感的限制性内切酶，更优选的，所述甲基化敏感的限制性内切酶选自HpaII或BstUI，或其组合。
(8)根据(6)所述的用途，其中，所述富集特征DNA所需的试剂选自杂交探针捕获法所需的试剂、聚合酶链式反应扩增法所需的试剂、锚定核酸扩增法所需的试剂、用于边合成边测序检测的试剂，或用于单分子测序的试剂，或其组合。
(9)根据(8)所述的用途，其中，所述富集特征DNA所需的试剂为探针或探针组；优选的，所述探针或探针组为EP-007探针或探针组。
(10)根据(1)-(9)任一项所述的用途，其中，其中，所述检测为产前检测；优选的，所述产前检测为无创产前检测；更优选的，所述产前检测为孕早期无创产前检测。
(11)根据(1)-(10)任一项所述的用途，其中，所述DNA为gDNA或cfDNA。
(12)一种试剂盒，其用于检测待测样品中至少一个特定的DNA甲基化修饰或特定的DNA甲基 化单倍型，以检测、监测或预测受试者是否患有胎盘源性妊娠期病症，所述试剂盒包含：
(a)检测DNA甲基化修饰的存在的试剂；
可选的，所述试剂盒还可以包含：
(b)检测DNA甲基化状态的显示剂；
其中，所述特定的DNA甲基化修饰或特定的DNA甲基化单倍型来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域。
(13)根据(12)所述的试剂盒，其中，所述的特定人类基因组区域如附表1所示的基因组区域；所述的特定的甲基化修饰为如附表1所述基因组区域来源DNA片段所携带的甲基化修饰。其中，所携带的甲基化修饰可以包含特定人类基因组区域的CpG位点的甲基化修饰或特定的DNA甲基化单倍型。所述甲基化差异区域为如附表1所示的基因组区域；
所述特定的DNA甲基化修饰为如附表1所示的基因组区域内特定CpG位点的甲基化修饰；
所述特定的甲基化单倍型为如附表1所示的基因组区域所覆盖的DNA甲基化单倍型；
所述特定的DNA甲基化修饰或特定的甲基化单倍型能区分正常妊娠孕妇胎盘和患有胎盘源性妊娠期病症的孕妇胎盘。
(14)根据(12)-(13)任一项所述的试剂盒，其中，所述检测DNA甲基化修饰的存在的试剂为富集特征DNA所需的试剂；优选的，所述富集特征DNA所需的试剂为探针或探针组；更优选的，所述探针或探针组为EP-007探针或探针组。
(15)根据(12)-(14)任一项所述的试剂盒，其中，所述DNA甲基化状态显示剂选自识别甲基化DNA的抗体或结合蛋白、亚硫酸氢盐、具有DNA催化氧化作用的酶类、具有DNA脱氨基作用的酶类或甲基化敏感酶，或其组合；优选的，所述甲基化敏感酶选自甲基化敏感的限制性内切酶，更优选的，所述甲基化敏感的限制性内切酶选自HpaII或BstUI，或其组合。
(16)根据(12)-(15)任一项所述的试剂盒，其中，所述待测样品选自来源于血液、尿液、粪便、唾液、口腔拭子、宫颈分泌物、宫颈涂片、羊水穿刺、胎儿绒毛或胎儿循环细胞的样品；优选的，所述血液为外周血，更优选的，所述外周血为血浆。
(17)根据(12)-(16)任一项所述的试剂盒，其中，所述检测为产前检测；优选的，所述产前检测为无创产前检测；更优选的，所述产前检测为孕早期无创产前检测。
(18)根据(12)-(17)任一项所述的试剂盒，其中，所述DNA为gDNA或cfDNA。
(19)一种多核苷酸，所述多核苷酸可以：
(i)在高严格性杂交条件或非常高严格性杂交条件下，与EP-007探针或探针组所示的多核苷酸杂交；或(ii)具有与(i)所示的多核苷酸的序列的反向互补序列；其中，所述多核苷酸用于检测样品中至少一个特定的DNA甲基化修饰或特定的DNA甲基化单倍型。
(20)如(19)所述的多核苷酸，其与EP-007探针或探针组所示的核苷酸序列或其反向互补序列具有至少90％，可选至少95％，优选至少97％，更优选至少98％，最优选至少99％的序列同一性的序列。
(21)如(19)或(20)所述的多核苷酸，其中，所述多核苷酸的序列包含如EP-007探针或探针组所示的核苷酸序列；优选的，所述多核苷酸的序列为如EP-007探针或探针组所示的核苷酸序列。
(22)如(19)或(20)所述的多核苷酸，其中，所述多核苷酸为DNA探针。
(23)一种检测待测样品中是否存在至少一个特定人类基因组区域上的，具有胎盘源性妊娠期病症特征的DNA片段的方法，所述方法包括检测步骤，所述检测步骤用于检测：
(a)待测样品中是否存在至少一种胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰或其修饰丰度；或
(b)待测样品中是否存在至少一种胎盘源性妊娠期病症特征的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度；
其中，所述胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰或所述DNA甲基化单倍型来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域。
(24)根据(23)所述的方法，其中，所述胎盘源性妊娠期病症特征的特定CpG位点的甲基化修 饰发生在如附表1所示的基因组区域所覆盖的位点上；
所述DNA甲基化单倍型为如附表1所示的基因组区域所覆盖的DNA甲基化单倍型。
(25)根据(23)-(24)任一项所述的方法，其中，所检测的所述特定的DNA甲基化修饰或特定的DNA甲基化单倍型位于选自如下(i)-(iii)所示的区域中的一个或多个区域：
(i)位于LTR12转座子家族区域；优选的，所述LTR12转座子为LTR12C转座子或LTR12E转座子；
(ii)位于FLT1基因及其调控区域；优选的，所述FLT1基因及其调控区域为人类染色体chr13:28800000bp-302000000bp之间的区域；
(iii)位于LIFR基因及其调控区域；优选的，所述LIFR基因及其调控区域为人类染色体chr5:38370000bp–38840000bp之间的区域。
(26)根据(23)-(25)任一项所述的方法，其通过检测DNA甲基化修饰的存在的试剂对所述(a)或(b)进行检测，其中，所述检测DNA甲基化修饰的存在的试剂为富集特征DNA所需的试剂；优选的，所述富集特征DNA所需的试剂为探针或探针组；更优选的，所述探针或探针组为EP-007探针或探针组。
(27)根据(23)-(26)任一项所述的方法，其中，所述方法还包括从待测样品中提取DNA的步骤，所述待测样品为外周血样品；优选的，所述外周血样品为血浆样品。
(28)根据(23)-(27)任一项所述的方法，其中，所述DNA为gDNA或cfDNA。
(29)一种检测、监测或预测受试者是否患有胎盘源性妊娠期病症的方法，所述方法包括下列步骤：
(1)检测步骤：
(a1)检测待测样品中是否存在至少一种胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰或其修饰丰度，所述胎盘源性妊娠期病症特征的DNA甲基化修饰来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域；或
(b1)检测待测样品中是否存在胎盘源性妊娠期病症特征的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度，所述DNA甲基化单倍型来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域；
(2)比较步骤：
(a2)比较所述待测样品的特定CpG位点的甲基化修饰或其修饰丰度的检测结果和具有或不具有胎盘源性妊娠期病症的胎盘样本的特定CpG位点的甲基化修饰或其修饰丰度的检测结果；或
(b2)比较所述待测样品的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度的检测结果和具有或不具有胎盘源性妊娠期病症的胎盘样本的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度的检测结果；
(3)判断步骤：
(i)如果(a2)或(b2)中，所述待测样品和不具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较低或相似度统计量值显著低于预期，或者所述待测样品和具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较高或相似度统计量值显著高于预期，那么所述受试者患有胎盘源性妊娠期病症；
(ii)如果(a2)或(b2)中，所述待测样品和不具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较高或相似度统计量值显著高于预期，或者所述待测样品和具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较低或相似度统计量值显著低于预期，那么所述受试者未患有胎盘源性妊娠期病症。
在一个具体的实施方式中，所述待测样品来源于所述受试者。
(30)根据(29)所述的方法，其中，所述胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰发生在如附表1所示的基因组区域所覆盖的位点上；所述DNA甲基化单倍型为如附表1所示的基因组区域所覆盖的DNA甲基化单倍型。
(31)根据(29)-(30)任一项所述的方法，其中，所检测的所述特定CpG位点的甲基化修饰或 特定的DNA甲基化单倍型位于选自如下(i)-(iii)所示的区域中的一个或多个区域：
(i)位于LTR12转座子家族区域；优选的，所述LTR12转座子为LTR12C转座子或LTR12E转座子；
(ii)位于FLT1基因及其调控区域；优选的，所述FLT1基因及其调控区域为人类染色体chr13:28800000bp-302000000bp之间的区域；
(iii)位于LIFR基因及其调控区域；优选的，所述LIFR基因及其调控区域为人类染色体chr5:38370000bp–38840000bp之间的区域。
(32)根据(29)-(31)任一项所述的方法，其中，所述胎盘源性妊娠期病症选自妊娠期糖尿病、双胎输血综合征、胎儿生长受限、妊娠期高血压、子痫前期、重度子痫前期、继发型子痫前期、非典型子痫前期、HELLP综合症中的一种或多种；优选的，所述妊娠期病症选自子痫前期、重度子痫前期、继发型子痫前期、非典型子痫前期中的一种或多种。
(33)根据(29)-(32)任一项所述的方法，其中，所述胎盘源性妊娠期病症的特异甲基化区域包括超甲基化区域和低甲基化区域。
(34)根据(29)-(33)任一项所述的方法，其中，步骤(3)中所采用的相似度统计量判断方法选自：相关分析(correlation)、t检验、Z检验、超几何检验、傅里叶分析(Fourier analysis)、小波分析(Wavelet analysis)、主成分分析(PCA)、流形嵌入(tSNE)、正交矩阵分解(NMF)、支持向量机(SVM)、K-最近邻(KNN)、K-均值(k-means)、线性回归(LM)、广义线性回归(GLM)、混合高斯模型(GMM)、神经网络(NN)、随机森林(RF)、自编码网络(Autoencoder)、深度神经网络(DNN)及其衍生变体。
(35)根据(29)-(34)任一项所述的方法，其中，通过检测DNA甲基化修饰的存在的试剂，检测特定CpG位点的甲基化修饰或其修饰丰度；或检测特定的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度。
(36)根据(35)所述的方法，其通过检测DNA甲基化修饰的存在的试剂进行检测，其中，所述检测DNA甲基化修饰的存在的试剂为富集特征DNA所需的试剂；优选的，所述富集特征DNA所需的试剂为探针或探针组；更优选的，所述探针或探针组为EP-007探针或探针组。
(37)根据(29)-(36)任一项所述的方法，其中，所述方法还包括从待测样品中提取DNA的步骤，所述待测样品为外周血样品；优选的，所述外周血样品为血浆样品。
(38)根据(37)所述的方法，其中，所述DNA为gDNA或cfDNA。
(39)根据(29)-(38)任一项所述的方法，其中，所述方法进一步包括富集所述DNA或对所述DNA进行测序的步骤。
(40)一种用于检测、监测和/或预测受试者是否患有胎盘源性妊娠期病症的设备，包括：
处理器；
用于存储处理器和执行指令的存储器；
其中，所述处理器被配制为在执行所述处理器可执行指令时，实现(29)-(38)任一项所述的方法。
(41)一种非易失性计算机可读存储介质，其上存储有计算机程序指令，所述计算机程序指令被处理器执行时，实现(29)-(38)任一项所述的方法。
(42)一种检测待测样品中至少一个特定CpG位点的特定甲基化修饰或至少一种DNA甲基化单倍型的系统，所述系统包括检测模块，所述检测模块用于检测：
(a)待测样品中是否存在至少一种胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰或其修饰丰度；或
(b)待测样品中是否存在至少一种胎盘源性妊娠期病症特征的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度；
其中，所述胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰或所述DNA甲基化单倍型来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域。
(43)根据(42)所述的方法，其中，所述胎盘源性妊娠期病症特征的特定CpG位点的甲基化修 饰发生在如附表1所示的基因组区域所覆盖的位点上；所述DNA甲基化单倍型为如附表1所示的基因组区域所覆盖的DNA甲基化单倍型。
(44)根据(42)-(43)任一项所述的系统，其中，所检测的所述特定的DNA甲基化修饰或特定的DNA甲基化单倍型位于选自如下(i)-(iii)所示的区域中的一个或多个区域：
(i)位于LTR12转座子家族区域；优选的，所述LTR12转座子为LTR12C转座子或LTR12E转座子；
(ii)位于FLT1基因及其调控区域；优选的，所述FLT1基因及其调控区域为人类染色体chr13:28800000bp-302000000bp之间的区域；
(iii)位于LIFR基因及其调控区域；优选的，所述LIFR基因及其调控区域为人类染色体chr5:38370000bp–38840000bp之间的区域。
(45)一种检测、监测和/或预测受试者是否患有胎盘源性妊娠期病症的系统，所述系统包括下列模块：
(1)检测模块，所述检测模块检测：
(a1)待测样品中是否存在至少一种胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰或其修饰丰度，所述胎盘源性妊娠期病症特征的DNA甲基化修饰来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域；或
(b1)待测样品中是否存在胎盘源性妊娠期病症特征的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度，所述DNA甲基化单倍型来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域；
(2)比较模块，所述比较模块比较：
(a2)比较所述待测样品的特定CpG位点的甲基化修饰或其修饰丰度的检测结果和具有或不具有胎盘源性妊娠期病症的胎盘样本的特定CpG位点的甲基化修饰或其修饰丰度的检测结果；或
(b2)比较所述待测样品的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度的检测结果和具有或不具有胎盘源性妊娠期病症的胎盘样本的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度的检测结果；
(3)判断模块，所述判断模块判断受试者是否患有胎盘源性妊娠期病症：
(i)如果(a2)或(b2)中，所述待测样品和不具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较低或相似度统计量值显著低于预期，或者所述待测样品和具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较高或相似度统计量值显著高于预期，那么所述受试者患有胎盘源性妊娠期病症；
(ii)如果(a2)或(b2)中，所述待测样品和不具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较高或相似度统计量值显著高于预期，或者所述待测样品和具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较低或相似度统计量值显著低于预期，那么所述受试者未患有胎盘源性妊娠期病症。
(46)根据(45)所述的系统，其中，所述胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰发生在如附表1所示的基因组区域所覆盖的位点上；所述DNA甲基化单倍型为如附表1所示的基因组区域所覆盖的DNA甲基化单倍型。
(47)根据(45)-(46)任一项所述的系统，其中，所检测的所述特定CpG位点的甲基化修饰或特定的DNA甲基化单倍型位于选自如下(i)-(iii)所示的区域中的一个或多个区域：
(i)位于LTR12转座子家族区域；优选的，所述LTR12转座子为LTR12C转座子或LTR12E转座子；
(ii)位于FLT1基因及其调控区域；优选的，所述FLT1基因及其调控区域为人类染色体chr13:28800000bp-302000000bp之间的区域；
(iii)位于LIFR基因及其调控区域；优选的，所述LIFR基因及其调控区域为人类染色体chr5:38370000bp–38840000bp之间的区域。
(48)根据(23)-(39)任一项所述的方法，其中，检测方法包含高通量测序。
(49)根据(23)-(39)任一项所述的方法，其中，检测方法包含定量PCR(qPCR)或数字PCR (digital PCR)。
(50)根据(23)-(39)任一项所述的方法，其中，检测方法包含甲基化特异PCR。
(51)根据(23)-(39)任一项所述的方法，其中，检测方法包含甲基化敏感酶切。
发明的效果
本公开的技术方案提供了一种检测待测样品中至少一种DNA甲基化修饰差异位点的甲基化修饰水平或至少一种DNA甲基化单倍型的方法、试剂盒以及相应的检测系统，其可以用于准确的体外筛查受试者是否患有胎盘源性妊娠期病症。
附图说明
图1示出了胎盘组织gDNA 0.8％琼脂糖凝胶电泳图。
图2示出了血浆中cfDNA片段大小分布。
图3示出了Tequila V3 DNA甲基化建库产物片段大小分布。
图4示出了经SeqCap EpiGiant探针捕获的文库片段大小。
图5示出了子痫前期胎盘与正常妊娠胎盘的差异甲基化区域。
图6示出了子痫前期特异的差异甲基化区域在全基因组上的分布图。
图7示出了LTR12C转座子富集子痫前期特异的超甲基化区域。
图8示出了人类精子特异的低甲基化区域的LTR12C的甲基化水平的改变伴随染色质开放程度的变化的示意图；其中，示例染色质的区域为chr1:212,028,092-212,163,353(GRCh37版本)。
图9示出了子痫前期特异的超甲基化区域附近的染色质可及性在8细胞期发生特异性的上升。
图10示出了子痫前期胎盘的新生甲基化缺陷。
图11示出了子痫前期胎盘在LIFR基因上的新生甲基化缺陷。
图12示出了子痫前期胎盘在FLT1基因调控区域上的新生甲基化缺陷。
图13示出了子痫前期特异的低甲基化区域附近的染色质可及性在囊胚和胎盘中上升。
图14示出了子痫前期特异的低甲基化附近的基因的表达水平在绒毛外滋养层细胞中显著上升。
图15示出了子痫前期特异的甲基化区域的DNA甲基化修饰水平可以区分不同发育阶段的胚胎(或组织)及不同的妊娠并发症。
图16示出了子痫前期特异的甲基化单倍型。
图17示出了EP-007探针或探针组的设计原理与验证。
图18示出了EP-007探针或探针组在不同杂交和洗脱温度组合下捕获后测序结果的在靶率和平均测序深度的比较结果。
图19示出了不同杂交时间对测序结果的在靶率的影响。
图20示出了经EP-007探针或探针组捕获的文库片段大小。
图21示出了训练用于预测子痫前期风险的模型的结果。
图22示出了子痫前期风险预测模型在测试集中灵敏度和特异性的表现结果。
图23示出了子痫前期风险预测模型可以区分不同严重程度的子痫前期亚型。
图24示出了子痫前期风险预测模型可以预测分娩过程中第一产程的最高收缩压。
具体实施方式
定义
当在权利要求和/或说明书中与术语“包含”联用时，词语“一(a)”或“一(an)”可以指“一个”，但也可以指“一个或多个”、“至少一个”以及“一个或多于一个”。
如在权利要求和说明书中所使用的，词语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。
在整个申请文件中，术语“约”表示：一个值包括测定该值所使用的装置或方法的误差的标准偏 差。
虽然所公开的内容支持术语“或”的定义仅为替代物以及“和/或”，但除非明确表示仅为替代物或替代物之间相互排斥外，本公开中的术语“或”是指“和/或”。
在本公开中，如无相反说明，本公开中的人染色体的位置编号，均基于GRCh37版本进行编号。
在本公开中，“胎儿”是指孕龄8周后的胚胎。“孕龄”是怀孕龄的量度，其中起点为女性的最后正常月经期(LMP)或其他方法估计的相应龄。在人产科学中，孕龄指胚胎或胎儿龄加两周。在人类中，尽管在37至42周内发生分娩是常见的，分娩通常发生在大约40周的孕龄。
在本公开中，“孕早期”是指孕龄在16周或之前。
在本公开中，“正常妊娠”是指在妊娠过程中未发生任何明显的妊娠并发症，包括但不限于妊娠期高血压、妊娠期糖尿病、双胎输血综合征、胎儿宫内生长受限、HELLP综合症、子痫前期、重度子痫前期、继发性子痫前期、非典型子痫前期、子痫。
在本公开中，“对照妊娠”是指无子痫前期、无重度子痫前期、无继发性子痫前期、无非典型子痫前期、无子痫的妊娠。也就是说，本公开中的“对照妊娠”可以包含“正常妊娠”。
在本公开中，“胎盘源性妊娠期病症”是指由于胎盘组织发育缺陷或功能缺失导致的妊娠过程中的病症。示例性的，本公开中的“胎盘源性妊娠期病症”选自妊娠期糖尿病、双胎输血综合征、胎儿宫内生长受限、妊娠期高血压、子痫、子痫前期、重度子痫前期、继发型子痫前期、非典型子痫前期或HELLP综合症。
在本公开中，“妊娠期糖尿病”是指妊娠前没有糖尿病的女性在怀孕时出现高血糖的症状。
在本公开中，“双胎输血综合征”是单绒毛膜双羊膜囊双胎中最常见的并发症，由于单绒毛膜双羊膜囊双胎共享一个胎盘，胎盘内存在大量的血管交通吻合，两个胎儿的血液直接沟通，一个胎儿为供血胎，发育迟缓、贫血、少尿、羊水过少、低血压；另一个胎儿为受血胎，多血质、尿多、羊水过多、心脏肥大、高血压、水肿。供血胎由于胎儿宫内发育窘迫，可能胎死宫内；受血胎也可由于心脏衰竭而死亡。
在本公开中，“胎儿生长受限”是指胎儿体重低于其孕龄平均体重第十百分位数或低于其平均体重的两个标准差。
在本公开中，“妊娠期高血压”是指孕20周后出现舒张压≥90mm Hg和(或)收缩压≥140mm Hg(间隔6小时到1周之间测定)且无蛋白尿，无神经系统症状，无肝肾损害症状，无HELLP综合症症状。
在本公开中，“子痫前期”是指妊娠20周后首次出现收缩压≥140mmHg和(或)舒张压≥90mmHg，尿蛋白≥300mg/24h或定性实验(+)。在本公开中，子痫前期可以包含重度子痫前期、继发型子痫前期、非典型子痫前期。
在本公开中，“重度子痫前期”是指妊娠20周后出现收缩压≥160mm Hg和(或)舒张压≥110mm Hg，24小时尿蛋白含量大于等于2.0g和(或)定性实验(++)以上，同时出现下列至少一项：血肌酐>106μmol/L或较前升高，血小板<100,000/mm 3或出现微血管溶血性贫血，乳酸脱氢酶或肝酶升高，伴有头痛或其他神经系统或视觉症状，持续性上腹不适或肺水肿。
在本公开中，“继发型子痫前期”是指妊娠前或孕龄20周前出现收缩压≥140mm Hg和(或)舒张压≥90mm Hg，并在妊娠过程中发生子痫前期或子痫。
在本公开中，“非典型子痫前期”是指出现妊娠期高血压症状，同时具备以下之一：神经系统症状，溶血，肝酶升高，血小板降低；但无蛋白尿(简略称之，即无肾损害症状的子痫前期)或出现蛋白尿(超过两次快速试纸阳性)，同时具备以下之一：神经系统症状，溶血，肝酶升高，血小板降低；但无高血压(简略称之，即无高血压症状的子痫前期)。
在本公开中，“HELLP综合症”是指妊娠期出现的以溶血、肝酶升高和血小板减少为特点的综合征，是妊娠期高血压疾病的严重并发症。
在本公开中，“转座子”又称可移动基因，跳跃基因，是一种可在基因组内插入和切离并能改变自身位置的DNA序列。
本公开中的“序列同一性”和“同一性百分比”指两个或更多个多核苷酸之间相同(即同一)的核苷酸的百分比。两个或更多个多核苷酸之间的序列同一性可通过以下方法测定：将多核苷酸的核苷酸序列进行比对且对经比对的多核苷酸中含有相同核苷酸的位置数目进行评分，且将其与经比对的多核苷酸中含有不同核苷酸的位置数目进行比较。多核苷酸可例如通过含有不同核苷酸(即取代或突变)或缺失核苷酸(即一个或两个多核苷酸中的核苷酸插入或核苷酸缺失)而在一个位置处不同。序列同一性可通过用含有相同核苷酸的位置数目除以多核苷酸的总数来计算。举例而言，可通过用含有相同核苷酸的位置数目除以多核苷酸中核苷酸的总数且乘以100来计算同一性百分比。
在一些技术方案中，本公开涉及用于限定两个多核苷酸互补程度的杂交条件严格性。可选的，前述多核苷酸可以选自DNA。本公开使用的“严格性”指杂交期间的温度和离子强度条件以及是否存在某些有机溶剂。严格性越高，靶核苷酸序列与经标记的多核苷酸序列之间的互补程度越高。“严格条件”指仅具有高频率互补碱基的核苷酸序列将杂交的温度和离子条件。本文使用的术语“在高严格性或非常高的严格性条件下杂交”描述了用于杂交和洗涤的条件。用于进行杂交反应的指导可见于Current Protocols in Molecular Biology，John Wiley和Sons，N.Y.(1989)，6.3.1-6.3.6中。
在本公开中，“高严格性杂交条件”是指，对于长度为至少100个核苷酸的探针而言，遵循标准DNA印迹程序，在42℃处在5X SSPE(saline sodium phosphate EDTA)、0.3％SDS、200微克/ml剪切并变性的鲑精DNA和50％甲酰胺中预杂交和杂交12至24小时。最后在65℃处使用2X SSC、0.2％SDS将载体材料洗涤三次，每次15分钟。
在本公开中，“非常高严格性杂交条件”是指，对于长度为至少100个核苷酸的探针而言，遵循标准DNA印迹程序，在42℃处在5X SSPE(saline sodium phosphate EDTA)、0.3％SDS、200微克/ml剪切并变性的鲑精DNA和50％甲酰胺中预杂交和杂交12至24小时。最后在70℃处使用2X SSC、0.2％SDS将载体材料洗涤三次，每次15分钟。
在本公开中，“全基因组亚硫酸氢盐测序法”又被称为WGBS(Whole-genome bisulfite sequencing)，其原理是用bisulfite处理待测样品，将基因组中未发生甲基化的C碱基转换成U，进行PCR扩增后变成T，与原本具有甲基化修饰的C碱基区分开来，再结合高通量测序技术，与参考序列比对，即可判断CpG/CHG/CHH位点是否发生甲基化，特别适用于绘制单碱基分辨率的全基因组DNA甲基化图谱。
在本公开中，“游离DNA”也被称为cfDNA，是存在于人的循环血、尿液及其他体液中的游离于细胞外的部分降解了的机体内源性DNA。cfDNA的长度大多为200bp以下，在血浆中以很低的浓度存在(约1-100ng/mL)。
在本公开中，“基因组DNA”也被称为“gDNA”，是指有机体在单倍体状态下的DNA全部含量。
在本公开中，“位点”对应于单个位点，其可以是单个碱基位置或一组相关碱基位置，例如CpG位点。
在本公开中，“DNA甲基化”是指生物体内在DNA甲基转移酶的催化下，以S-腺苷甲硫氨酸(SAM)为甲基供体，将甲基转移到特定的碱基上的过程。在哺乳动物体内，主要是核苷酸胞嘧啶残基的5’C端的甲基化。在人类基因组上，大量的DNA甲基化发生在CpG双核苷酸中的胞嘧啶上，C是胞嘧啶，G是鸟嘌呤，p是磷酸基团。DNA甲基化也可以发生在CHG和CHH等核苷酸序列的胞嘧啶中，其中H是腺嘌呤、胞嘧啶或胸腺嘧啶。DNA甲基化还可以发生在非胞嘧啶上，例如N6-甲基腺嘌呤。此外，DNA甲基化还可以是5-羟甲基胞嘧啶等形式。在大部分情况下，DNA甲基化为对侧DNA链或DNA碱基附近其它的CpG位点上的甲基化修饰所诱导产生。
在某些情况下，“DNA甲基化”可以作为名词使用，指被甲基化后的DNA片段。
在本公开中，“去甲基化”是指原有的DNA片段上CpG位点的C碱基上的甲基化修饰被生物化学过程擦除。在人类胚胎发育过程中，该擦除过程主要是通过TET家族氧化酶特异地催化氧化5-甲基胞嘧啶依次成为5-羟甲基胞嘧啶、5-醛甲基胞嘧啶、5-羰甲基胞嘧啶，进而通过APOBEC家族酶类的催化脱氨基反应形成尿嘧啶，最后通过DNA聚合酶复制更正将该位点的尿嘧啶擦除，更换为原本的胞嘧啶。
在本公开中，“新生甲基化”是指原本无任何甲基化修饰的一段DNA片段上，发生的DNA甲基 化过程。特别地，该过程与半保留复制引致的对侧DNA链上甲基化修饰诱导生成的DNA甲基化并不相同。
在本公开中，“超甲基化”是指DNA片段上的CpG位点上的C碱基超过50％，优选地超过90％，更优选地超过95％被甲基化修饰。在本公开中，“超甲基化”与“高甲基化”含义相同，可互相替代。
在本公开中，“低甲基化”是指DNA片段上的CpG位点上的C碱基少于50％，优选地少于10％，更优选地少于5％被甲基化修饰。
在本公开中，“DNA甲基化位点”是指可能发生DNA甲基化修饰的单个或多个碱基位置。例如CpG位点、CHG位点或CHH位点。在一些情况下，DNA甲基化位点等同于CpG位点。
在本公开中，“DNA差异甲基化位点”也可以被称为“DNA甲基化修饰差异位点”，是指DNA甲基化修饰程度在两组生物样品中存在差异的单个碱基位置。例如CpG位点、CHG位点或CHH位点。
在本公开中，“DNA差异甲基化区域”也可以被称为“DNA甲基化差异区域”或“DNA甲基化修饰差异区域”，是指DNA甲基化修饰程度在两组生物样品中存在差异的多个近邻碱基位置所构成的基因组区域。其中，任意两个近邻碱基位置之间的距离不超过20000个核苷酸(碱基)。
在本公开中，“DNA甲基化修饰”可以被表述为单碱基甲基化修饰，也可被表述为一个连续区域上的多个碱基甲基化修饰的平均值，也可被表述为甲基化单倍型，也可被表述为甲基化单体型。
在本公开中，“DNA甲基化修饰水平”可用整体的定量参数β或甲基化单倍型的丰度来描述。
在本公开中，DNA甲基化修饰水平整体的定量参数β可表示为，C碱基的平均甲基化修饰程度，即：
Figure PCTCN2020120654-appb-000001
其中，上述公式无论对于单个碱基或多个近邻碱基所构成的基因组区域皆成立。
在本公开中，“甲基化单倍型”也被称为“甲基化单体型”，其是指单条DNA片段上，连续两个或多个C碱基位点的甲基化修饰的组合。由于DNA甲基化修饰在同一条染色体(DNA链)上存在相关性，因此，可能存在多个邻近的C碱基位点，其甲基化修饰情况相互关联，形成一定的固定形式。因此，DNA甲基化单倍型为DNA甲基化修饰在同一条染色体上存在的连锁模式。
在本公开中，“甲基化单倍型的丰度”，是指对甲基化单倍型信息进行统计分析后，得到特定甲基化单倍型所占的比例。即：甲基化单倍型的丰度＝符合特定甲基化单倍型的DNA片段数/完全覆盖该甲基化单倍型基因组区域的DNA片段总数。
在本公开中，“胎盘源性妊娠期病症的甲基化区域”和“胎盘源性妊娠期病症胎盘特异的差异甲基化区域(DMRs)”所表示的含义相同，可以相互替换。
在本公开中，“Pearson相关系数”可以用于度量两个随机变量的相关程度，其在协方差的基础上除以了两个随机变量的标准差。Pearson相关系数是一个介于-1和1之间的值，当两个变量的线性关系增强时，相关系数趋于1或-1；当一个变量增大，另一个变量也增大时，表明它们之间是正相关的，相关系数大于0；如果一个变量增大，另一个变量却减小，表明它们之间是负相关的，相关系数小于0；如果相关系数等于0，表明它们之间不存在线性相关关系。
在本公开中，“相似度”可以用于度量两个样品或两类样品的定量相似程度。特别地，其可以被定量地表示为通过统计学原理统计后，得出的一个样品的确来自某一类特定样品的概率。换言之，其相当于从特定的一类样品组中随机抽取一个样品，发现该样品等价于给定的目标样品的概率。
在本公开中，“相似度较高”或者“显著高于”是指当某个样品来自A类特定样品而非来自其它类特定样品(例如非来自于B类特定样品)的概率超过90％，优选地，超过95％，更优选地，超过99％，即，相似度统计量值显著高于预期；“相似度较低”或“显著低于”是指当某个样品来自A类特定样品而非来自其它类特定样品(例如非来自于B类特定样品)的概率低于10％，优选地，低于5％，更优选地，低于1％，即，相似度统计量值显著低于预期。
在本公开中，度量两样品间相似程度的方法，可以选自贝叶斯方法，相关性分析，二项分布，beta分布，负二项分布，超几何分布，正态分布，泊松分布，Komolgorov-Smirnov检验(KS检验)，Shapiro-Wilk检验。度量样品相似程度之比(即，某个样品更可能来自A类特定样品而非B类特定样品的概率)的方法，可以选自似然比检验，卡方检验。不失一般性地，本领域技术人员应容易认识到，基于本公开提供的特异的DNA甲基化标志物及统计原理方法，可以使用其他任何适当的数理统计方法，进行该类分析。
在本公开中，“EP-007探针或探针组”是由一系列长度约为50-70bp的，末端被标记的DNA片段构成，其能够捕获富集本公开附表1所示的基因组区域。针对前述附表1中所示的一个特定的区域，EP-007探针或探针组中包含这一特定区域所有CpG位点的全甲基化或全去甲基化的修饰组合，即对前述附表1中所示任一区域的任一CpG位点，EP-007探针或探针组既包含该位点为非甲基化胞嘧啶情况下的互补序列，又包含该位点为甲基化胞嘧啶情况下的互补序列。示例性的，本公开中“EP-007探针或探针组”中的探针的长度为50，51，52，53，54，55，56，57，58，59，60，61，62，63，64，65，66，67，68，69或70bp。
在本公开的一个实施方式中，可以使用其他技术对DNA甲基化状态进行分析，例如微阵列、实时PCR、数字实时PCR和质谱分析等(Plongthongkum et al.,Nature Reviews Genetics,2014,15(10):647-61；Consortium et al.,Nature Biotechnology,2016,34(7):726)。本领域技术人员应容易认识到，基于本公开提供的特异的DNA甲基化标记物，使用任何可以检测DNA甲基化状态的方法或试剂均可检测、监测和/或预测受试者是否患有胎盘源性妊娠期病症。
基于亚硫酸氢盐转化原理的甲基化检测方法，已经成为检测DNA甲基化状态的常规技术，而且也是本领域技术人员公知的。在本公开的一个具体实施方案中，采用来自Zymo Research公司的EZ DNA Methylation-Gold亚硫酸氢盐转化试剂盒作为检测DNA甲基化状态的试剂，对样品DNA进行亚硫酸氢盐转化。
在本公开的一个实施方式中，发明人发现，可以通过将待测胎盘样品与对照妊娠的胎盘的甲基化区域进行比较，通过比较待测胎盘样品和具有或不具有特定胎盘源性妊娠期病症孕妇来源的胎盘样本，判断待测样本在特定胎盘源性妊娠期病症胎盘特异的差异甲基化区域的平均甲基化水平或甲基化单倍型丰度，从而通过该平均甲基化水平或甲基化单倍型丰度分析待测胎盘样品与具有或不具有特定胎盘源性妊娠期病症胎盘的相似度，进而判断待测胎盘样品的来源是否和特定胎盘源性妊娠期病症有关。
在本公开的一个具体的实施方式中，发明人发现，具有胎盘源性妊娠期病症胎盘与对照妊娠的胎盘相比较，存在一系列胎盘源性妊娠期病症胎盘特异的差异甲基化区域(DMRs)，即胎盘源性妊娠期病症的甲基化区域。
在本公开的另一个实施方式中，发明人发现，可以通过比较胎盘源性妊娠期病症胎盘特异的差异甲基化区域(DMRs)的平均甲基化水平，进而区分正常妊娠胎盘与胎盘源性妊娠期病症孕妇的胎盘，所述胎盘源性妊娠期病症选自妊娠期糖尿病(GDM)、双胎输血综合征(TTTS)、胎儿宫内生长受限、妊娠期高血压(GHT)、子痫、子痫前期(PE)、重度子痫前期、继发型子痫前期、非典型子痫前期或HELLP综合症患者的胎盘，进而判断受试者是否存在胎盘源性妊娠期病症。
在本公开的一个具体的实施方式中，胎盘源性妊娠期病症为子痫前期。示例性的，子痫前期胎盘特异的差异甲基化区域(PE-DMRs)按其相对甲基化水平，即在相同区域内与对照妊娠胎盘的区域甲基化水平进行比较，可分为子痫前期特异的超甲基化区域(PE-hyper-DMRs，即在子痫前期胎盘中甲基化修饰水平增高的基因组区域)和子痫前期特异的低甲基化区域(PE-hypo-DMRs，即在子痫前期胎盘中甲基化修饰水平降低的基因组区域)。进一步地，发明人发现，通过比较PE-DMRs区域的平均甲基化水平可以区分不同发育阶段的生殖细胞或胚胎组织，包括但不限于卵子、精子、8细胞卵裂期、桑椹胚、囊胚期内细胞团、孕6周的胎儿等。
在本公开的一个实施方式中，通过进一步研究PE-DMRs，发明人发现PE-hyper-DMRs多分布于基因间区域和内含子。进一步地，发明人发现一系列位于基因间或内含子上的人类内源逆转录病毒(endoretrovirus，ERV)上存在较高比例的PE-hyper-DMRs。
在本公开的一个具体的实施方式中，富集子痫前期特异超甲基化的ERV包括LTR12C、LTR12E、LTR13A、HERVE-int、LTR12D、LTR13等。在一个更具体的实施方式中，显著富集子痫前期特异的PE-hyper-DMRs的ERV是LTR12C。
尽管不希望受单一理论约束的限制，存在PE-hyper-DMRs区域在人类(Homo sapiens)精子细胞中处于低甲基化(hypo-methylation)状态但在黑猩猩(Pan troglodytes)中的同源区域上处于高甲基化(hyper-methylation)状态，即PE-hyper-DMRs富集于人类配子特异的低甲基化区域。更进一步地，PE-hyper-DMRs区域的染色质可及性在人类胚胎发育8细胞期发生特异性的上升，即PE-hyper-DMRs富集于在胚胎发育特定时期起重要功能的基因组功能区域。
在本公开的一个实施方式中，发明人发现，子痫前期胎盘中，LTR12C甲基化状态的改变与其周围基因的染色质可及性状态改变强烈相关。
另一方面，PE-hypo-DMRs在功能基因的内含子、非编码RNA的内含子、5’非翻译区、3’非翻译区和基因下游区域富集。
在本公开的一个实施方式中，发明人发现，PE-hypo-DMRs显著地富集在胎盘谱系新生甲基化区域，其表明子痫前期胎盘在新生甲基化的过程中存在明显缺陷。进一步地，PE-hypo-DMRs区域的染色质可及性在人类胚胎发育桑椹胚期、囊胚期及胎盘中发生特异的上升，且其附近的基因表达在该胚胎发育阶段特别是在绒毛外滋养层细胞(EVT)的分化过程中强烈上升。即PE-hypo-DMRs富集于在细胞分化及胎盘发育过程中起重要功能的基因组功能区域。
在本公开的另一个实施方式中，发明人发现，胎盘组织中，子痫前期特异的甲基化区域中存在有较强相关性的CpG位点。该类基因组区域来源的DNA片段上携带的甲基化修饰可形成特征性的组合，即子痫前期特异的甲基化单倍型。进一步地，发明人发现子痫前期特异的甲基化单倍型的甲基化模式在子痫前期胎盘和对照妊娠胎盘中存在明显且稳定的差异。同时，这类甲基化单倍型在正常未孕女性的血浆中不存在。
在本公开的另一个实施方式中，发明人发现，在且仅在罹患子痫前期的孕妇血浆中的cfDNA片段可检测出子痫前期特异的甲基化单倍型。
在本公开的另一个实施方式中，发明人发现，孕妇血浆中这类甲基化单倍型的丰度差异，实际上指代了胎盘发育健康状况的差异。也就是说，可以通过比较受试孕妇血浆和具有胎盘源性妊娠期病症胎盘及对照妊娠胎盘中的甲基化单倍型的丰度差异，判断受试孕妇是否患有胎盘源性疾病。
在本公开的一个具体实施方式中，当血浆中子痫前期特异的甲基化单倍型丰度在统计学意义上显著较子痫前期孕妇血浆中的丰度低，对照妊娠特异的甲基化单倍型丰度在统计学意义上显著较子痫前期孕妇血浆中的丰度高时，可认为胎盘发育健康状况良好；而子痫前期特异的甲基化单倍型丰度相对升高时，可认为胎盘发育不良，更可能发生子痫前期等胎盘源性疾病。因此，发明人发现的子痫前期特异甲基化单倍型，可用于预测子痫前期风险。
在本公开的一个实施方式中，发明人发现，可以通过比较所述待测样品和具有或不具有所述胎盘源性妊娠期病症的胎盘样本，比较待测样品在所述具有胎盘源性妊娠期病症胎盘特异的差异甲基化区域的平均甲基化水平；进而通过比较所述待测样品和不具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度，判断所述受试者是否患有胎盘源性妊娠期病症。
具体的，如果检测结果相似度较低(即相似度统计量值显著低于预期)，或者所述待测样品和具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较高(即相似度统计量值显著高于预期)，那么所述受试者患有胎盘源性妊娠期病症；如果所述待测样品和不具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较高(即相似度统计量值显著高于预期)，或者所述待测样品和具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较低(即相似度统计量值显著低于预期)，那么所述受试者未患有胎盘源性妊娠期病症。
其中，所述相似度较高(或显著高于)或较低(或显著低于)的判断标准为：如果该样本来自某一组所述样本的概率低于10％，优选地低于5％，更优选地低于1％，则认为检测结果相似度较低；如果该样本来自某一组所述样本的概率高于90％，优选地高于95％，更优选地高于99％，则认为检测结 果相似度较高。
在本公开的另一个实施方式中，发明人发现，可以通过比较所述待测样品的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度的检测结果和具有或不具有胎盘源性妊娠期病症的胎盘样本的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度的检测结果，进而判断所述受试者是否患有胎盘源性妊娠期病症。
具体的，如果所述待测样品和不具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较低(即相似度统计量值显著低于预期)，或者所述待测样品和具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较高(即相似度统计量值显著高于预期)，那么所述受试者患有胎盘源性妊娠期病症；如果所述待测样品和不具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较高(即相似度统计量值显著高于预期)，或者所述待测样品和具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较低(即相似度统计量值显著低于预期)，那么所述受试者未患有胎盘源性妊娠期病症。
其中，所述相似度较高(或显著高于)或较低(或显著低于)的判断标准为：如果该样本来自某一组所述样本的概率低于10％，优选地低于5％，更优选地低于1％，则认为检测结果相似度较低；如果该样本来自某一组所述样本的概率高于90％，优选地高于95％，更优选地高于99％，则认为检测结果相似度较高。
在本公开的一个实施方式中，发明人利用22例孕11-18周的孕妇血浆建立了对子痫前期风险的预测模型。具体地，提取孕妇血浆中cfDNA，通过特定探针捕获亚硫酸盐处理后的cfDNA片段，富集含子痫前期特异的甲基化区域的DNA片段，进行高通量测序。通过计算该cfDNA样品与子痫前期和对照妊娠胎盘的相似度。
在本公开的一个实施方式中，可以通过计算cfDNA样品与任一类胎盘样品之间的Pearson相关系数，对子痫前期的风险进行预测。
在本公开的一个具体的实施方式中，Pearson相关系数可以用于描述受试孕妇cfDNA中的子痫前期特异的甲基化单倍型与对照妊娠胎盘和子痫前期胎盘的相关程度，即r(CTRL)和r(PE)。进一步地，通过比较r(CTRL)和r(PE)而对受试孕妇在该孕程罹患子痫前期的风险进行评估。r(CTRL)>r(PE)，受试孕妇在本次妊娠中患子痫前期的风险为低风险；r(CTRL)<r(PE)，受试孕妇在本次妊娠中患子痫前期的风险为高风险；r(CTRL)＝r(PE)，受试孕妇在本次妊娠中患子痫前期的风险为灰度临界值。
进一步地，在本公开的一个回顾性临床试验中，对于159例孕妇外周血样品，该检测和统计方法对子痫前期的孕妇的检出率达到了97％的灵敏度及89％的特异性。进一步地，该相关系数也可以预测孕妇在本孕程中罹患妊娠期高血压、子痫前期、重度子痫前期和继发性子痫前期的风险。进一步地，该相关系数与孕妇在第一产程的最高收缩压血压呈良好的线性相关，并可通过该相关系数对生产过程的最高收缩压血压进行预测。
在本公开的一个具体的实施方式中，“EP-007探针或探针组”由一系列长度为50-70bp的3’端被生物素标记的DNA片段构成，其序列包括以下四类序列(其设计原理如本公开的图17的a部分所示)：
1.与附表1中所示的基因组区域的序列中，C更换为T后的序列反向互补的序列；
2.附表1中所示的基因组区域的序列中，C更换为T后的序列；
3.与附表1中所示的基因组区域的序列中，除CpG位点以外的C更换为T后的序列反向互补的序列；
4.附表1中所示的基因组区域的序列中，除CpG位点以外的C更换为T后的序列。
在本公开的一个具体的实施方式中，所述标记可以选自在3’末端被标记。
在本公开的一个具体的实施方式中，所述标记可以包括但不限于生物素标记、亲和素标记、荧光标记等标记方式，只要所述标记可以使得所述“EP-007探针或探针组”被检测仪器检测到即可。示例性的，如果采用荧光标记，那么检测仪器选自能够检测荧光的设备。
由于本公开中附表1中所示的基因组区域的序列已经被记载，因此，本领域技术人员可以依据上述EP-007探针或探针组的设计原理，进而设计合适的EP-007探针或探针组。
实施例
本公开的其他目的、特征和优点将从以下详细描述中变得明显。但是，应当理解的是，详细描述和具体实施例(虽然表示本公开的具体实施方案)仅为解释性目的而给出，因为在阅读该详细说明后，在本公开的精神和范围内所作出的各种改变和修饰，对于本领域技术人员来说将变得显而易见。
本公开中采用的分子生物学方法，均可以参见“最新分子生物学实验方法汇编(Current Protocols in Molecular Biology，Wiley出版)”，“分子克隆实验指南(Molecular Cloning：A Laboratory Manual，冷泉港实验室出版)”等公开出版物中记载的相应方法。
本实施例中采用的所有试剂，除非另有强调，否则均可以通过商业途径购买获得。
本公开涉及的内容，可利用任何一种适当的生物材料实施，文中实施例仅为示例使用。领域内的专业工作者不难使用其他生物材料或其中若干种的组合，包括但不限于：血液、尿液、粪便、唾液、口腔拭子、宫颈分泌物、宫颈涂片、羊水穿刺样品、胎儿绒毛样品、胎儿循环细胞等材料，实现本公开所涉及的检测。
本公开涉及的实验内容，可利用任何一种适当的DNA甲基化修饰测定方法实施，文中实施例仅为示例使用。领域内的专业工作者不难使用其他方法或其中若干种的组合，包括但不限于：亚硫酸盐转化、酶促甲基化修饰转化、Sanger测序、定量PCR、数字PCR、飞行时间质谱、甲基化特异PCR反应、甲基化特异限制性酶切、简并甲基化测序、全基因组甲基化测序、杂交捕获测序、扩增子测序、反向PCR(Inverse PCR)、斑点印记、生物芯片杂交、单分子测序、纳米孔测序、滚环扩增放大(LAMP)、CRISPR酶联放大反应、抗体交联酶联放大反应等方法，实现本公开所涉及的检测。
本公开涉及的检测后分析，可利用任何一种适当的数学分析方法实施，文中实施例仅为示例使用。领域内的专业工作者不难使用其他方法或其中若干种方法的组合，包括但不限于：t检验、Z检验、超几何检验、傅里叶分析(Fourier analysis)、小波分析(Wavelet analysis)、主成分分析(PCA)、流形嵌入(tSNE)、正交矩阵分解(NMF)、支持向量机(SVM)、K-最近邻(KNN)、K-均值(k-means)、线性回归(LM)、广义线性回归(GLM)、混合高斯模型(GMM)、神经网络(NN)、随机森林(RF)、自编码网络(Autoencoder)、深度神经网络(DNN)及其衍生变体等方法，实现本公开所涉及的分析。
实施例1：利用Qiagen试剂盒对孕妇胎盘组织gDNA进行提取
实验材料
冷冻胎盘组织(收集自广东省妇幼保健院，临床研究批准号：医伦第[201701044]号)；
无水乙醇(北京化工，00500)；
Qiagen动物组织提取gDNA试剂盒(Qiagen，69504)；
Qubit TM dsDNA HS Assay Kit(Thermo Fisher Scientific，Q32851)；
λDNA HindIII digest(Takara,3403)
实验方法
胎盘组织消化：取约25mg胎盘组织并配制消化液(180μL缓冲液ALT+20μL蛋白酶K)，将其混合后置于65℃孵育4小时至完全消化。
细胞裂解：将消化后产物涡旋后加入裂解液缓冲液AL、无水乙醇及RNaseA并将其在室温孵育10分钟。
gDNA结合：将孵育后的全部样品转移至吸附柱中，离心，弃废液。
清洗及洗脱：依次用缓冲液AW1和缓冲液AW2对吸附膜进行清洗后加入洗脱液提取出gDNA。
实验结果
如图1所示，图1中左起第一泳道为DNA分子标记物，即经Hind III酶切的λDNA产物(Takara)。DNA分子标记物的最上面的条带大小为23,130bp。从左起第二泳道至第六泳道为从不同胎盘组织中提取的gDNA。与此同时，图1中的电泳条带分布无明显的弥散状，表明其完整性良好。
通过实施例1记载的技术方案，用约25mg胎盘组织提取出8640ng gDNA。经Qubit TM dsDNA HS  Assay Kit测定，其平均浓度约为86.4ng/μL，图中gDNA条带集中在约20kb，表明片段大小约为20kb。
实施例2：利用MagMAX游离DNA提取试剂盒对孕妇血浆中cfDNA进行提取
实验材料
存储于南科征途MiniMax TM游离DNA(cfDNA)无创管(Apostle,Inc.Silicon Valley,California.)、Cell-Free DNA BCT CE采血管(Streck,Inc.,La Vista,NE)等采血管的外周血(收集自广东省妇幼保健院，临床研究批准号：医伦第[201701044]号)；
蛋白酶K(Qiagen，19133)；
SDS(Sigma，74255-250G)；
Dynabeads TMMyOne TM Silane(Thermo Fisher Scientific，37002D)；
MagMAX TM Cell Free DNA Lysis/Binding Solution(Thermo Fisher Scientific，A33600)；
MagMAX TM Cell Free DNA Wash Solution(Thermo Fisher Scientific，A33601)；
无水乙醇(北京化工，00500)；
Qubit TM dsDNA HS Assay Kit(Thermo Fisher Scientific，Q32851)；
EB缓冲液(Qiagen，19086)；
DNase/RNase-free水(索莱宝，R1600)。
实验方法
样品准备：将装有外周血的游离DNA采血管放入离心机中离心使其分层并分离血浆层。
血浆裂解：配制裂解反应液(含0.4mg/ml蛋白酶K、血浆和1％SDS)将其涡旋、离心并在60℃条件下孵育20分钟。孵育结束后，将裂解反应液冷却至室温。
游离DNA与磁珠结合：按MyOne磁珠：Binding Solution为3:250的比例配制结合预混液，将其与血浆裂解产物充分混合涡旋震荡。
磁珠的清洗：将反应管放置于磁力架上吸附后，用Wash Solution清洗磁珠两次后再用80％无水乙醇清洗两次。清洗磁珠后移除无水乙醇，保持反应管于磁力架上，开盖静置干燥。
游离DNA的洗脱：待磁珠干燥后加入EB缓冲液洗脱并转移至新的离心管中储存。
实验结果
用2mL血浆提出总量10ng cfDNA，其浓度为0.5ng/μL(用Qubit TM dsDNA HS Assay Kit测定)。样品中cfDNA片段大小的分布由Agilent 2100生物分析仪测定。
图2中所示为Agilent2100生物分析仪测定的cfDNA的片段大小分布。其中，在35bp和10,380bp出现的峰为DNA分子标记。cfDNA的主峰出现在约161bp。
如图2所示，提取出的cfDNA的样品无gDNA污染，其片段大小分布的主峰在161bp，基本与理论值一致。
实施例3：利用Tequila V3 DNA甲基化建库方法对gDNA或cfDNA建库
实验材料
7.5-10ng cfDNA或200ng gDNA；
EZ DNA Methylation-Gold TM Kit(ZymoReserch,D5006)；
Tequila V3 DNA甲基化建库试剂盒(优乐复生，EU-TQ-Methy)；
Agencourt AMPure XP磁珠(Beckman，A63882)；
EB缓冲液(Qiagen，19086)；
DEPC水(索莱宝，R1600-500)；
无水乙醇(北京化工，00500)。
实验方法
亚硫酸盐转化：依照EZ DNA Methylation-Gold TM Kit试剂盒使用说明书配制CT转化试剂并对上述起始量的cfDNA或gDNA进行CT转化处理。其中，前述cfDNA或gDNA通过实施例2所述的方法得到。
末端修复：用Tequila V3 DNA甲基化建库试剂盒，依照表1配制末端修复反应混合物1。其对应的PCR反应程序：37℃ 30min，95℃ 10min，热盖105℃，体积20μL。
表1修复反应液试剂加量
反应体系 单个样品用量μL
cfDNA或gDNA 17
TQRB1缓冲液 2
TQRE1酶 1
总体积 20
连接反应1：用Tequila V3 DNA甲基化建库试剂盒，按照表2在冰上配制反应混合物2。其对应的PCR反应程序：37℃ 30min，95℃ 5min，10℃保持，热盖105℃，体积30μL。
表2连接反应1试剂加量
反应体系 单个样品用量μL
上一步反应 20
NF‐H 2O 1.5
TQLB1缓冲液 4
TQLR1试剂 2
TQLR2试剂 1.5
TQLE1酶 1
总体积 30
线性扩增：用Tequila V3 DNA甲基化建库试剂盒，按照表3配制反应混合物3。其对应的PCR反应程序：95℃,3min，12X【95℃ 30s；60℃ 30s；68℃ 1min】，68℃ 5min，16℃保持，热盖105℃，体积100μL。
表3 线性扩增反应液加量
反应体系 单个样品用量μL
上一步反应 30
DEPC水 45
TQAB1 20
TQAR1 2
TQAR2 2
TQAE1 1
总体积 100
磁珠纯化1：用Tequila V3 DNA甲基化建库试剂盒，138μL TQSB试剂与27μL XP磁珠充分混匀后加入线性扩增的PCR产物，经室温孵育后上磁力架吸附。吸附至液体澄清后，移除上清，加入180μL TQSB试剂与100μL DEPC水的混合物将磁珠重悬。经室温孵育后上磁力架吸附，吸附至澄清后，移除上清，用80％乙醇清洗磁珠后加入洗脱液。
DNA变性：PCR程序为95℃ 10min，热盖105℃，体积10μL。当程序运行5min后，立即将装样品的0.2mL PCR管从PCR仪中取出，立即插于冰上，放置2min。
连接反应2:用Tequila V3 DNA甲基化建库试剂盒，按照表4在冰上配制反应混合物4，其PCR反应程序为25℃15min，热盖35℃，体积20μL。
表4连接反应2试剂用量
反应体系 单个样品用量μL
上一步产物 6.6
TQTHB1缓冲液 10
TQTHR1试剂 2.4
TQHE1酶 1
总体积 20
磁珠纯化2:向上一部反应产物中加入17μL XP磁珠，并将其充分涡旋混匀后于室温静置孵育5min。孵育后上磁力架吸附至液体澄清后，弃掉上清。用80％乙醇清洗磁珠两次后，将磁珠在室温干燥并用DEPC水洗脱。
PCR扩增：PCR扩增体系如表5所示，相应的反应程序为98℃ 45s，12X【98℃ 15s，60℃ 30s，72℃ 30s】，72℃ 5min，10℃保持，热盖105℃，体积100μL。
表5 PCR扩增体系
反应体系 单个样品用量μL
上一步反应洗脱液 73
TQAB1试剂 20
TQAR1试剂 2
TQIE2酶 1
Index T5XX 2(需单独加)
Index Q7XX 2(需单独加)
总体积 100
磁珠纯化3:向上一步PCR产物中加入17μL XP磁珠，充分混匀后放于室温静置孵育5min后上磁力架吸附至液体澄清。吸弃废液后用80％乙醇清洗磁珠两次。吸弃废液后将磁珠在室温干燥后加入DPEC水洗脱，获得预文库。
实验结果
实验结果如图3所示，其示出了Agilent2100生物分析仪测定的cfDNA的片段大小分布。其中，在35bp和10,380bp出现的峰为DNA分子标记。
以10ng cfDNA的起始量经该实验方法得出61.8ng/μL的预文库(用Qubit TM dsDNA HS Assay Kit测定)，其片段大小分布的主峰约为320bp，即其中cfDNA预文库的片段大小约为320bp(参见图3中的a部分)。
以200ng胎盘gDNA为起始量可得70.8ng/μL的预文库(用Qubit TM dsDNA HS Assay Kit测定)，其片段大小分布的主峰约为300bp，即其中gDNA预文库的片段大小约为300bp(参见图3中的b部分)。
实施例4：通过SeqCap EpiGiant探针对gDNA/cfDNA全基因组甲基化位点富集
实验材料
甲基化建库的预文库产物500ng；
无创子痫前期早筛试剂盒的杂交洗脱试剂盒(优乐复生，EU-TQ-MV1)；
SeqCap EpiGiant富集探针(Roche，07138911001)；
Agencourt AMPure XP磁珠(Beckman，A63882)；
Dynabeads TM M-270链霉亲和素(Thermo Fisher ScientificScientific，65306)。
实验方法
蒸干：用无创子痫前期早筛试剂盒的杂交洗脱试剂盒，按表6配制蒸干体系于1.5mL EP管中并放置于真空浓缩仪中开盖60℃蒸干。其中，文库样品按照实施例3所记载的方法构建。
表6蒸干体系
反应体系 单个组分用量μL
文库样品 500ng
TQBLKC试剂 2
TQBLKD试剂 4
TQBLKCHE试剂 2.5
杂交反应：用无创子痫前期早筛试剂盒的杂交洗脱试剂盒，按表7配制杂交体系于200μL PCR管中，涡旋混匀后于室温静置10min。其后在PCR仪中进行变性，95℃ 10min。待变性8min时将装有4.5μL探针的PCR管放入95℃的PCR仪上，预热探针。变性结束后，在47℃下将探针加入杂交体系混合物中。涡旋混合后，放置于47℃ PCR仪(热盖温度为57℃)杂交反应72小时。
表7杂交体系
反应体系 单个样品用量μL
上一步蒸干样品 /
TQHS1 7
TQHS2 3.5
捕获清洗：取室温平衡过的链霉素修饰的磁珠(Dynabeads TM M-270链霉亲和素)100μL、200μL磁珠洗涤缓冲液(1X TQWS5试剂)与上一步反应产物充分混合后置于磁力架上吸附至液体澄清。吸弃液体，用200μL磁珠洗涤缓冲液清洗磁珠一次后用等体积磁珠洗涤缓冲液将磁珠重悬，置于磁力架上吸附至液体澄清。吸弃废液后置于PCR仪中孵育45min。
洗脱：用无创子痫前期早筛试剂盒的杂交洗脱试剂盒，在上述步骤产物中加入180μL经65℃预热的1X TQWS4并于65℃震荡孵育5min后置于磁力架上吸附至液体澄清，重复一次。吸弃废液，加入180μL 1X TQWS1于常温震荡混匀2min后置于磁力架上吸附至液体澄清。吸弃废液，加入180μL 1X TQWS2于常温震荡混匀1min后置于磁力架上吸附至液体澄清。吸弃废液，加入180μL 1X TQWS3于常温震荡混匀1min后置于磁力架上吸附至液体澄清。吸弃废液，加入23μL TQNF试剂震荡混匀。
捕获产物的扩增：用无创子痫前期早筛试剂盒的杂交洗脱试剂盒，按表8配制捕获产物扩增体系，其相应的PCR反应程序为98℃ 45s，8X【98℃ 15s；60℃ 30s；72℃ 30s】72℃ 2min,16℃保持，热盖105℃，体积50μL。
表8捕获产物扩增体系
反应体系 单个样品用量μL
TQAE2酶 25
TQPR3试剂 2
捕获产物(含磁珠) 23
总体积 50
扩增后纯化：用无创子痫前期早筛试剂盒的杂交洗脱试剂盒，将上述反应产物置于磁力架上吸附至液体澄清后吸取上清与90μL XP磁珠涡旋混合。室温静置5min后置于磁力架上吸附至液体澄清。吸弃废液，用80％乙醇清洗两次磁珠后，于室温开盖晾干。待磁珠干燥后加入25μL TQEB试剂于室温孵育5min后上磁力架吸附至液体澄清。吸取澄清液体到新的EP管中即为纯化后的捕获产物。
实验结果
以500ng甲基化建库的预文库起始，由Qubit TM dsDNA HS Assay Kit测定经SeqCap EpiGiant探针捕获后可富集4.46ng/μL文库。
图4所示的是Agilent 2100生物分析仪测定SeqCap EpiGiant探针捕获文库的片段大小，前述片段的大小约为300bp。具体来说，图4中的a部分所示为胎盘gDNA所建文库，其片段大小约为300bp。图4中的b部分所示为血浆中cfDNA所建文库，其片段大小约为300bp。
实施例5：甲基化文库Illumina平台二代高通量测序后数据的质量检测
实验方法和结果
DNA预文库按照Illumina平台试剂盒说明书上机测序。测序下机数据中，Q30比例大于80％。拆分样品使用bcl2fastq(0.2.1)。质控标准未知拆分片段数<5％，平均片段长度>160bp。PhiX文库使用Euler-PhiX流程质控，合格标准为error rate<0.001％。分文库使用Fastqc(3.2)进行质控。文库质控标准为：Q30>80％，重复片段<＝10％，GC比例为30％-35％。对于游离DNA文库，片段长度分布与标准游离NA片段长度分布KS检验P>0.5，片段末端碱基分布与标准游离DNA片段末端碱基分布KS检验P>0.5。抽样CHG>THG、CHH>THH变换比例≥99％。
实施例6：子痫前期胎盘特异的差异甲基化区域
实验材料
对照妊娠胎盘(收集自北京大学附属第三医院，临床研究伦理审查批件号：IRB00001052-16009)；
子痫前期孕妇胎盘(收集自北京大学附属第三医院，临床研究伦理审查批件号：IRB00001052-16009)；
人类精子细胞DNA甲基化数据库(CRA000114)；
黑猩猩精子细胞DNA甲基化数据库(GSE30340)；
人类胚胎2细胞期ATAC-seq数据库(SRP163205、SRP112718)；
人类胚胎4细胞期ATAC-seq数据库(SRP163205、SRP112718)；
人类胚胎8细胞期ATAC-seq数据库(SRP163205、SRP112718)；
人类胚胎囊胚期内细胞团ATAC-seq数据库(SRP163205、SRP112718)；
人类胚胎囊胚期滋胚层ATAC-seq数据库(SRP163205、SRP112718)；
人类胚胎干细胞ATAC-seq数据库(SRP112718)；
HBSS(Thermo Fisher Scientific,14025076)；
蛋白酶抑制剂(Roche,5056489001)；
0.4％台酚蓝(索莱宝,C0040)；
60％Iodixanol(Sigma,D1556)；
TruePrep DNA Library Prep Kit(Vazyme,TD501-02)；
TruePrep Index Kit V2 for Illumina(Vazyme,TD202)；
Oligo Clean&Concentrator(Zymo Research,D4061)；
Agencourt AMPure XP磁珠(Beckman，A63882)；
RNase-free水(索莱宝,R1600-500)；
70um细胞筛(BD Falcon,352350)。
实验方法
DNA甲基化水平分析
胎盘gDNA提取，进行SeqCap EpiGiant探针捕获和高通量测序的流程同实施例1，实施例4和实施例5。
CpG位点甲基化水平分析：使用PileOMeth(版本号：0.1.13)提取各个CpG位点上测序深度及甲基化水平。质控条件：要求单个CpG位点上测序深度≥200X。使用R语言(3.5.2)对每个甲基化位点进行分析，筛选子痫前期特异的甲基化修饰位点，使用t检验对每个CpG甲基化修饰位点进行统计检测，要求其服从以下条件：其在子痫前期胎盘样品中甲基化修饰程度的分布与对照胎盘样品中的甲基化修饰程度分布存在显著差异，p<0.001。同时，要求其在子痫前期胎盘样品中甲基化修饰程度的分布与健康未孕女性血浆样品中的甲基化修饰程度的分布存在显著差异，p<0.001。通过上述分析得到的CpG位点的甲基化水平，在不同组样品之间，即子痫前期胎盘、对照妊娠胎盘和未孕女性血浆组存在显著且稳定的甲基化水平的差异，上述分析得到的CpG位点为子痫前期胎盘特异的甲基化修饰位点，详见附表1。
对子痫前期胎盘特异的甲基化修饰位点所在的区域进行合并，对整个区域内的甲基化水平进行平均，在对照胎盘和子痫前期胎盘中，使用R语言(3.5.2)对每个区域进行分析，使用t检验对每个区域中的平均甲基化水平进行检测。
染色质可及性测序分析(ATAC-seq)实验流程
组织匀浆：取20mg冷冻的组织于预冷的玻璃匀浆器中，加入1ml预冷的1x HB缓冲液(如表10所示，其中表10所示的1x HB缓冲液通过表9所示的1.022xHB缓冲液进一步配制)后对组织进行研磨。将研磨后的组织匀浆液过70微米的细胞筛并转移至低吸附的管子中，4℃离心5min。弃上清，用新鲜HB缓冲液重悬。
表9 1.022xHB缓冲液的配制
Figure PCTCN2020120654-appb-000002
表10 1xHB缓冲液的配制
Figure PCTCN2020120654-appb-000003
碘克沙醇(Iodixanol)梯度离心：向重悬液中加入等体积的50％碘克沙醇，配制30％及40％碘克沙醇并构建25％/30％/40％碘克沙醇富集细胞核的密度梯度离心体系，4℃ 3000rcf离心20min。离心后吸取位于30％和40％碘克沙醇交界面的细胞核层。加入RSB缓冲液(表11)对细胞核进行稀释。
表11 RSB缓冲液的配制
Figure PCTCN2020120654-appb-000004
Tn5转座酶酶切建库及纯化：4℃ 500rcf离心10min后，依照VazymeTD501-02 TruePrepDNALibraryPrepKit试剂盒的说明书进行Tn5转座酶酶切。依照Zymo D4061 Oligo Clean&Concentrator试剂盒对酶切产物进行纯化。依照TruePrep Index Kit V2 for Illumina试剂盒对纯化产物进行Index PCR。对PCR产物用XP磁珠进行文库纯化。
ATAC‐seq数据分析
使用deeptools(版本号3.3.0)提取覆盖度，使用R软件(版本号3.5.2)分析转录因子及TSS区覆盖值，使用macs2软件(版本号2.2.4)及Genrich软件(版本号0.6)分析开放染色质富集区域峰。对子痫胎盘及对照胎盘抽取的开放染色质富集区域峰，利用diffBind及NarrowPeaks(R语言包来源于Bioconductor)分析其差异区域并进行富集分析(使用R语言进行数据统计分析)。
实验结果
子痫前期特异DNA甲基化区域的筛选
如图5中的a部分所示，横轴表示正常妊娠胎盘在胎盘特异的差异甲基化区域的平均甲基化水平，纵轴表示子痫前期胎盘在相同区域的平均甲基化水平。浅灰色点集中分布于直线y＝x，表示这些甲基化区域的平均甲基化程度在子痫前期和正常妊娠胎盘中一致，即为无差异的区域；黑色点分布明显偏离直线y＝x，其代表在子痫前期和正常妊娠胎盘中有显著统计学差异的差异甲基化区域。
图5b中密度分布曲线描述的是图5a的差异甲基化区域。因此，如图5中的b部分所示，其图5的a部分中无差异区域(浅灰)和有显著统计学差异的甲基化区域(深灰)的密度分布曲线。
也就是说，如图5所示，通过比较子痫前期和对照妊娠胎盘在全基因范围内的甲基化水平发现存 在大量子痫前期特异的差异甲基化区域(PE-DMRs)。按PE-DMRs甲基化水平的不同可将其分为子痫前期特异的超甲基化区域(PE-hyper-DMRs)和子痫前期特异的低甲基化区域(PE-hypo-DMRs)，详见附表2。
如图6所示，将PE-DMRs比对到全基因上发现，PE-DMRs分布集中在基因调控区域，如非编码转录区(5’UTR/3’UTR)，基因间区域(intergenic region)，内含子(intron)以及部分外显子区域。进一步的统计富集分析表明，PE-hypo-DMRs显著地富集在内含子、5’UTR/3’UTR和非编码RNA的内含子区域；而虽然PE-hyper-DMRs多分布于基因间区域和内含子区域，其分布模式是随机的。
如图7所示，将PE-hyper-DMRs区域与人类基因组中转座子元件(transposable element，TE)相比对发现，LTR12家族转座子显著的富集了PE-hyper-DMRs。特别地，约30％的LTR12C转座子包含PE-hyper-DMRs。
如图8中的a部分所示，其为黑猩猩和人类精子细胞的DNA甲基化模式；如图8中的b部分所示，其为对照妊娠和子痫前期胎盘的染色质可及性的比较；如图8中的c部分所示，其为对照妊娠和子痫前期胎盘中该区域上甲基化模式。
也就是说，比较子痫前期胎盘、对照妊娠胎盘、人类精子细胞和黑猩猩精子细胞在具有子痫前期特异的超甲基化特征的LTR12C区域或其同源区域的DNA甲基化修饰水平发现，存在位于子痫前期超甲基化区域的LTR12C，该区域在黑猩猩精子细胞中为超甲基化状态而在人类精子细胞中为低甲基化状态(图8中的a部分)，即存在人类精子特异的低甲基化的LTR12C在子痫前期胎盘中处于超甲基化的状态(图8中的c部分)。人类精子特异的低甲基化修饰的LTR12C在子痫前期胎盘中DNA甲基化状态发生改变并伴随着染色质可及性的变化(图8中的b部分)
如图9所示，分析胚胎不同发育时期(2细胞期、4细胞期、8细胞期、囊胚期内细胞团、胚胎干细胞和胎盘)的全基因组染色质可及性发现，子痫前期超甲基化区域附近的基因的染色质可及性在8细胞期特异性的升高。
实施例7：胎盘谱系特异的新生甲基化区域的筛选
实验方法和结果
CpG位点甲基化水平分析：使用PileOMeth(版本号0.1.13)提取各个CpG位点上测序深度和甲基化水平。质控条件：要求单个CpG位点≥200X。使用R语言(3.5.2)对每个甲基化位点进行分析，筛选胎盘特异的超甲基化修饰位点且该位点在健康未孕女性白细胞中处于低甲基化状态。使用t检验对每个CpG甲基化修饰位点进行统计检测，要求其服从的条件：其在对照妊娠胎盘样品中甲基化修饰程度的分布与健康未孕女性白细胞中样品中的甲基化修饰程度分布存在显著差异，p<0.001。
通过前述分析筛选出的差异修饰的甲基化位点将其区域进行合并，对整个区域内的甲基化水平进行平均，进而筛选出胎盘特异的超甲基化区域。类似地，比较HUVEC(一种已分化的人源胎盘细胞系)和hESC1(一种未分化的人源胚胎干细胞系)DNA甲基化差异修饰的位点可以筛选出在HUVEC中超甲基化且在hESC1中低甲基化的位点，使用t检验对差异甲基化位点进行统计检验，须满足p<0.001。将筛选出的差异修饰的甲基化位点进行区域合并，并对整个区域内的甲基化水平进行平均，进而筛选出HUVEC特异的超甲基化区域。将胎盘特异的超甲基化区域与HUVEC特异的超甲基化区域取交集即为胎盘谱系特异的新生甲基化区域。
实施例8：子痫前期特异的低甲基化区域富集在胎盘谱系新生甲基化区域
实验材料
对照妊娠胎盘(收集自北京大学附属第三医院，临床研究伦理审查批件号：IRB00001052-16009)；
子痫前期孕妇胎盘(收集自北京大学附属第三医院，临床研究伦理审查批件号：IRB00001052-16009)；
人类胚胎2细胞期ATAC-seq数据库(SRP163205、SRP112718)；
人类胚胎4细胞期ATAC-seq数据库(SRP163205、SRP112718)；
人类胚胎8细胞期ATAC-seq数据库(SRP163205、SRP112718)；
人类胚胎囊胚期内细胞团ATAC-seq数据库(SRP163205、SRP112718)；
人类胚胎干细胞ATAC-seq数据库(SRP112718)；
人类胚胎囊胚期滋胚层ATAC-seq数据库(SRP163205、SRP112718)；
人类胎盘组织单细胞转录组学测序数据库(E-MTAB-6701、E-MTAB-6678、E-MTAB-7304)
实验方法和结果
对胎盘新生甲基化区域与PE-DMRs取交集，分选出子痫前期特异的差异甲基化且参与胎盘新生甲基化的区域，即ExE-DN PE-DMRs和子痫前期特异的差异甲基化但不参与胎盘新生甲基化的区域，即Not-ExE-DN PE-DMRs。
如图9所示，子痫前期特异的差异甲基化且参与胎盘新生甲基化的区域，ExE-DN PE-DMR(深灰色)和子痫前期特异的差异甲基化但不参与胎盘新生甲基化的区域，Not-ExE-DN PE-DMR(浅灰色)的区域平均甲基化水平，其中纵轴代表子痫前期胎盘与正常妊娠胎盘的区域平均甲基化水平的比值的log值，即在y＝0代表子痫前期胎盘与正常妊娠胎盘在某区域的平均甲基化水平无差异。
也就是说，与对照妊娠胎盘相比，子痫前期胎盘在ExE-DN PE-DMRs区域的甲基化水平显著降低(图10)。将子痫前期特异的低甲基化区域与胎盘新生甲基化区域进行比对发现52％子痫前期特异的低甲基化区域与胎盘新生甲基化区域重叠，故子痫前期胎盘在胎盘谱系新生甲基化过程中存在明显缺陷。两个子痫前期特异的胎盘谱系新生甲基化缺陷实例如图11和图12所示。分析胚胎不同发育时期(2细胞期、4细胞期、8细胞期、囊胚期内细胞团、胚胎干细胞和胎盘)的全基因组染色质可及性发现，在子痫前期特异的低甲基化区域附近的基因的染色质可及性在桑椹胚、囊胚或胎盘中特异性的升高，例如图13所示。
单细胞RNA表达时间序列分析：使用Monocle(版本号0.2.0)提取RNA表达数据，使用主成分分析方法(reduce_dimension函数，Monocle)及UMAP方法对数据进行降维。对每个细胞，计算其同族(PE-hypo-DMR或PE-hyper-DMR位置附近50kbp碱基对范围内的基因)基因的表达量平均值，作为PE-hypo-DMR基因和PE-hyper-DMR基因的表达量。
通过分析胎盘绒毛细胞细胞滋养细胞(VCT)分化成合体细胞滋养细胞(SCT)或绒毛外滋养细胞(EVT)过程中基因表达水平发现，如图14所示，子痫前期特异的低甲基化区域附近的基因多在EVT细胞中特异表达而子痫前期特异的超甲基化区域附近的基因多在VCT细胞中特异表达。
实施例9：子痫前期特异的甲基化区域的平均甲基化水平可以区分不同发育阶段的生殖细胞或胚 胎组织
实验材料
人类卵子DNA甲基化数据库(CRA000114)
人类精子DNA甲基化数据库(CRA000114)
人类胚胎8细胞卵裂期DNA甲基化数据库(CRA000114)
人类胚胎桑椹胚期DNA甲基化数据库(CRA000114)
人类胚胎囊胚期内细胞团组织DNA甲基化数据库(CRA000114)
人类6孕周胎儿DNA甲基化数据库(CRA000114)
子痫前期孕妇胎盘(收集自北京大学附属第三医院，临床研究伦理审查批件号：IRB00001052-16009)
正常妊娠胎盘(收集自北京大学附属第三医院，临床研究伦理审查批件号：IRB00001052-16009)
实验方法和结果
对胎盘进行DNA甲基化测序实验方法和数据分析同实施例4、实施例5和实施例6。
通过上述实施例的实验和分析可得出PE-hyper-DMRs和PE-hypo-DMRs区域。对人类卵子、精子、8细胞卵裂期、桑椹胚、囊胚期内细胞团和6孕周胎儿的甲基化数据库中在上述区域的平均甲基化水平进行统计发现，配子结合后的早期胚胎发育过程包含一个广泛的全基因组去甲基化(以8细胞卵裂期、 桑椹胚、囊胚为代表)再到新生甲基化(以正常胎盘和胚胎为代表)的过程。在该表观遗传学变化过程的轨迹上，以子痫前期特异的超甲基化区域和低甲基化区域上的平均甲基化水平可将子痫前期胎盘和早期胚胎/胎儿进行良好的区分。
图15中的a部分和b部分分别为在子痫前期特异的超甲基化和低甲基化区域上，不同发育阶段的胚胎(或组织)、不同妊娠并发症的胎盘的平均甲基化水平。图15中的c部分为以在子痫前期特异的超甲基化区域的平均甲基化水平为横坐标、以在子痫前期特异的低甲基化区域的平均甲基化水平为纵坐标对不同发育阶段的生殖细胞或胚胎组织绘制散点图，子痫前期胎盘、不具有胎盘源性妊娠期病症的胎盘、精子、卵子、8细胞期、桑椹胚、囊胚可被良好地区分。
也就是说，如图15所示，该结果进一步印证了PE-hyper-DMRs和PE-hypo-DMRs为子痫前期胎盘特异。
实施例10：子痫前期特异的甲基化区域的平均甲基化水平可以区分不同妊娠并发症的胎盘
实验材料
妊娠期高血压孕妇胎盘
妊娠期糖尿病孕妇胎盘
双胎输血综合征孕妇胎盘
子痫前期胎盘
正常妊娠胎盘
(以上胎盘样品均收集自北京大学附属第三医院，临床研究伦理审查批件号：IRB00001052-16009)
实验方法和结果
对子痫前期、妊娠期高血压、妊娠期糖尿病、双胎输血综合征、正常妊娠孕妇胎盘进行DNA甲基化测序实验方法和数据分析同实施例4和实施例6。
通过上述实施例中的实验和分析可得出PE-hyper-DMRs和PE-hypo-DMRs区域，对妊娠期高血压、妊娠期糖尿病、双胎输血综合征、不具有胎盘源性妊娠期病症的胎盘和子痫前期孕妇胎盘在上述区域内的平均甲基化水平进行统计发现，子痫前期特异的超甲基化区域和低甲基化区域的平均甲基化水平可以区分不同妊娠并发症(图15c)，故PE-hyper-DMRs和PE-hypo-DMRs在子痫前期胎盘中存在特异的甲基化修饰差异。
实施例11：子痫前期组和对照妊娠组的甲基化单倍型的区别
实验材料
对照妊娠胎盘
子痫前期孕妇胎盘
健康未孕女性血浆
对照妊娠孕妇血浆
子痫前期孕妇血浆
(以上样品均收集自广东省妇幼保健院，临床研究批准号医伦第[201701044]号)
实验方法和结果
胎盘组织gDNA/血浆cfDNA的提取、DNA文库的构建流程大致同实施例1、实施例2、实施例4和实施例5。
对前述分析得到的子痫前期特异的差异甲基化区域内进行甲基化单倍型抽提，使用R语言(3.5.2)对每个单倍型进行分析，筛选子痫前期特异的甲基化单倍型，要求其满足在子痫前期胎盘中的出现的频率至少为在对照胎盘中出现频率的20倍；或满足其在对照胎盘中出现的频率至少是在子痫前期胎盘中出现频率的20倍。
筛选出的甲基化单倍型详见附表3，该表中所列的子痫前期特异的甲基化单倍型在对照妊娠胎盘和子痫前期胎盘中频率存在显著而稳定的差异，例如图16所示。其中，图16的a部分和b部分分别示例 子痫前期特异的超甲基化区域和低甲基化区域的甲基化单倍型。
实施例12：EP-007探针或探针组的设计与验证
实验材料
合成的EP-007探针或探针组；
KAPA双链DNA建库试剂盒(KAPA HTP Lib PrepKit，KK8235)；
Agencourt AMPure XP磁珠(Beckman，A63882)；
Dynabeads TM M-270链霉亲和素(Thermo Fisher Scientific，65306)。
实验方法与结果
如图17中的a部分所示，EP-007探针或探针组设计的基本原理如下：该探针由若干条长度为59bp的与经亚硫酸盐测序后的甲基化单倍型特异位点序列互补的序列组成。对每个子痫前期特异的甲基化单倍型，其中的每个的CpG位点将合成对应该位点的甲基化(G)和非甲基化(A)的探针各一条。覆盖相邻位点的两条探针含有40bp的相互重叠序列。在探针的3’端有生物素标记物。
通过前述分析得到的子痫前期特异甲基化单倍型(附表3)，对其所在的基因组区域，进行探针设计，合成与其互补的DNA探针(Twist Inc.，美国)。如图17中的a部分所示，其基本设计为59bp长度的DNA互补链。
示例性的，在本实施例中，希望检测的目标区域的序列为：
>13:29140769-29140827
Figure PCTCN2020120654-appb-000005
该序列为FLT1基因上游区域的一部分。基于本公开记载的探针的设计原理，针对前述区域设计的探针为：
1.5’-TAGGTAATTTGTAAAGGAGGGTTAATTGCGATTGTTTGGTTTATTTTTAGAGATTAGAA-3’(即CpG->C；非CpG->T)(SEQ ID NO：2)
2.5’-TAGGTAATTTGTAAAGGAGGGTTAATTGTGATTGTTTGGTTTATTTTTAGAGATTAGAA-3’(即所有C->T)(SEQ ID NO：3)
3.5’-GTTCGTTGGATATTTTTTTTCGGTTGACGTTGACGGATCGGGTGAGGGTTTTTGGTTTT-3’(即反向互补。CpG->C；非CpG->T)(SEQ ID NO：4)
4.5’-GTTTGTTGGATATTTTTTTTTGGTTGATGTTGATGGATTGGGTGAGGGTTTTTGGTTTT-3’(即反向互补。所有C->T))(SEQ ID NO：5)
对合成后的DNA探针，按照KAPA双链DNA建库试剂盒的操作说明书，对其进行双链建库测序。如表12所示，小于1X测序深度的数据所占比几乎为0且标准化的平均测序深度在0.2以下的几乎为0，故该探针可良好地覆盖所选区域。
表12 EP-007探针或探针组双链建库的测序结果
Figure PCTCN2020120654-appb-000006
与此同时，如图17中的b部分所示，对EP-007探针或探针组进行双链建库后的测序结果。横坐标为不同探针片段的测序深度与平均测序深度之比，纵坐标代表分布密度。该测序深度分布，符合泊松分布。因此，EP-007探针或探针组的合成质量是无偏、可靠的。
实施例13：优化EP-007探针或探针组的杂交时间及杂交和洗脱的反应温度
实验材料
甲基化建库的预文库产物500ng；
无创子痫前期早筛试剂盒的杂交洗脱试剂盒(优乐复生，EU-TQ-MV1)；；
Agencourt AMPure XP磁珠(Beckman，A63882)；
Dynabeads TM M-270链霉亲和素(Thermo Fisher Scientific，65306)。
实验方法
大致同实施例4
实验结果
杂交和洗脱温度
图18中的a部分和b部分，分别示出了在不同杂交温度(47℃、56℃和65℃)和洗脱温度(47℃、56℃和65℃)组合下的测序结果的在靶率和平均测序深度的比较。其中，圆形为在47℃下杂交，方形为在56℃下杂交，三角形为在65℃下杂交。
通过比较在不同杂交温度(47℃、56℃和65℃)和洗脱温度(47℃、56℃和65℃)组合下文库产物的测序结果的在靶率(on target ratio)和平均测序深度(mean coverage)得出在56℃杂交，65℃洗脱捕获的效果最优，即在靶率大于30％(如图18中的a部分所示)且在相同数据量下平均测序深度最高(如图18中的b部分所示)。
杂交时间
图19示出了在杂交时，56℃的洗脱温度下不同杂交时间(4h、8h、12h、24h、36h、48h和60h)的文库产物的在靶率发现杂交时间达12h即可达到在靶率大于30％的测序结果。
从结果来看，杂交时间在12h以上可获得大于30％的在靶率的测序结果。
实施例14：通过EP-007探针或探针组对gDNA或cfDNA子痫前期的甲基化修饰差异位点进行捕获
实验材料
甲基化建库的预文库产物500ng；
无创子痫前期早筛试剂盒的杂交洗脱试剂盒(优乐复生，EU-TQ-MV1)；；
Agencourt AMPure XP磁珠(Beckman A63882)；
Dynabeads TM M-270链霉亲和素(Thermo Fisher Scientific，65306)。
实验方法
大致同实施例4
实验结果
图20示出了Agilent2100生物分析仪测定的经探针捕获后的文库的片段大小分布。在35bp和10,380bp出现的峰为DNA分子标记。其中，图20的a部分示出了通过胎盘gDNA所建文库的片段大小，图15的b部分示出了通过血浆中cfDNA所建文库的片段大小。
也就是说，以500ng甲基化建库的预文库起始，经EP-007探针或探针组捕获可富集2.98ng/μL文库。通过Agilent 2100生物分析仪测定，EP-007探针或探针组捕获的，通过胎盘gDNA所建文库的片段大小和通过血浆中cfDNA所建文库的片段大小均约为300bp。
实施例15：子痫前期风险的预测模型的构建
实验材料
对照及子痫前期孕妇于孕周10-38周之间抽取的，存储于南科征途MiniMax TM游离DNA(cfDNA)无创采血管(Apostle,Inc.Silicon Valley,California)的外周血；(收集自广东省妇幼保健院，临床研究批准号医伦第[201701044]号)
蛋白酶K(Qiagen，19133)；
SDS(Sigma，74255-250G)；
Dynabeads TMMyOne TM Silane(Thermo Fisher Scientific，37002D)；
MagMAX TM Cell Free DNA Lysis/Binding Solution(Thermo Fisher Scientific，A33600)；
MagMAX TM Cell Free DNA Wash Solution(Thermo Fisher Scientific，A33601)；
无水乙醇(北京化工，00500)；
EB缓冲液(Qiagen，19086)；
DNase/RNase-free水(索莱宝，R1600)；
EZ DNA Methylation-Gold TM Kit(Zymo Research,D5006)；
Tequila V3 DNA甲基化建库试剂盒(优乐复生，EU-TQ-Methy)；
EB缓冲液(Qiagen，19086)；
SeqCap EpiGiant富集探针(Roche，07138911001)；
Agencourt AMPure XP磁珠(Beckman，A63882)；
Dynabeads TM M-270链霉亲和素(Thermo Fisher Scientific，65306)；
ILLUMINA NextSeq PE150测序试剂盒。
实验方法和结果
对22例孕妇的血浆进行分子生物学实验及DNA甲基化水平分析，实验方法同实施例2、3、5、9和12。其中包含6例对照妊娠孕妇，16例子痫前期孕妇。
在对照妊娠和子痫前期组，对附表3中每个子痫前期特异的甲基化单倍型区域的甲基化水平在组内取平均值而得到矩阵，M(CTRL)和M(PE)，通过缺一交叉验证法(leave-one-out)，计算得出与对照妊娠和子痫前期的Pearson相关系数r(CTRL)和r(PE)。
如图21所示，将r(CTRL)和r(PE)与22例孕妇的临床诊断做线性回归分析可得到线性分数，可用来评估子痫前期相关的胎盘发育不良的程度，即罹患子痫前期的孕妇胎盘发育不良程度高，相应的，其线性分数高；而对照妊娠的孕妇胎盘发育相对良好，故其线性分数低。
如图16所示，其示出了对PE-DMRs区域甲基化水平分析经相关性分析和线性回归统计计算的线性分数。
实施例16：回顾性临床样品验证子痫前期风险预测模型
实验材料
同实施例13，其中，共使用159名患子痫前期或对照妊娠孕妇于10-38孕周抽取的外周血标本(广东省妇幼保健院医伦第[201701044]号)。
实验方法和结果
分四批次，由四个实验人员对159例孕妇的血浆进行分子生物学实验及单倍型分析，实验方法同实施例2、3、5、9和12。
对附表3中的子痫前期特异的甲基化单倍型区域，计算受试者在每区域的平均甲基化水平而得到矩阵M(test)，分别计算M(test)和M(CTRL)和M(PE)的Pearson相关系数，即r(CTRL)和r(PE)。进而比较r(CTRL)和r(PE)而实现对子痫前期的风险进行预测，若r(CTRL)>r(PE)，子痫前期高风险；r(CTRL)<r(PE)，子痫前期低风险。
如图22所示，结果显示，该方法可区分子痫前期孕妇与对照妊娠孕妇，其灵敏度为97％(36/37)，特异性为89％(109/122)。
实施例17：子痫前期风险预测模型可区分不同严重程度的子痫前期亚型
实验材料
同实施例13，其中，共使用159名患子痫前期或正常孕妇于10-38孕周抽取的外周血标本(收集自广东省妇幼保健院，临床研究批准号医伦第[201701044]号)
实验方法和结果
实验方法大致同实施例15。对于159例受试孕妇血浆，对r(CTRL)和r(PE)及其临床结局做相关性分析发现可将妊娠期高血压、子痫前期、重度子痫前期和继发性子痫前期较好地区分(图23)。
实施例18：子痫前期风险预测模型可以预测孕妇生产时第一产程的最高压
实验材料
同实施例13，其中，共使用181名患子痫前期或对照妊娠孕妇于10-38孕周抽取的外周血标本(收集自广东省妇幼保健院，临床研究批准号医伦第[201701044]号)
实验方法和结果
首先对22例孕妇的r(CTRL)和r(PE)和其对应的临床结局中第一产程的最高压做回归统计分析，建立受试胎盘与对照妊娠和子痫前期胎盘的相似程度与孕妇第一产程的最高收缩压之间的模型。然后，利用该模型对159例孕妇第一产程的最高收缩压进行预测。如图24所示，该模型预测出的第一产程最高收缩压与临床测得的最高收缩压呈良好的线性关系。
本公开的上述实施例仅是为清楚地说明本公开所作的举例，而并非是对本公开的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本公开的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本公开权利要求的保护范围之内。
附表1
1号染色体基因组区域：
10609-10732；854755-855015；855281-855548；865578-865745；906938-907140；918299-918633；933296-933610；981271-981434；1002867-1003145；1064840-1065000；1091548-1091808；1096501-1096908；1098952-1099212；1100360-1100496；1103155-1103312；1104187-1104346；1141776-1142016；1145641-1145909；1222110-1222220；1267412-1267789；1268092-1268275；1268747-1268985；1269212-1269412；1353574-1353851；1356727-1356902；1377431-1377557；1487465-1487644；1713644-1713847；1906534-1906862；1910292-1910489；1953059-1953419；2064041-2064202；2066274-2066422；2124985-2125125；2160972-2161105；2161785-2162095；2164535-2164639；2262419-2262565；2270684-2270893；2279479-2279833；2307620-2307890；2316165-2316792；2339934-2340225；2350002-2350312；2365498-2365658；2379747-2379888；2382636-2382868；2387845-2387952；2391121-2391407；2401568-2401734；2403576-2403735；2408494-2408653；2409799-2410059；2411377-2411499；2415770-2416030；2425732-2426018；2435099-2435257；2437018-2437230；2500798-2500922；2527240-2527344；2719832-2719954；2722273-2722421；2766756-2766872；2774240-2774347；2777158-2777281；2779044-2779211；2782993-2783117；2792661-2792830；2799264-2799432；2827786-2827930；2835104-2835267；2835823-2835958；2836448-2836736；2844082-2844430；2844871-2845339；2845494-2845639；2846855-2846999；2850500-2850638；2850819-2850952；2859522-2859835；2883701-2883805；2886654-2886923；2899449-2899656；2918366-2918705；2919692-2919797；2938885-2939081；2980518-2980846；3006206-3006330；3007620-3007769；3026100-3026234；3032651-3032868；3042333-3042541；3044643-3045005；3051474-3051627；3057365-3057479；3059136-3059331；3059717-3059866；3079075-3079335；3080079-3080462；3080672-3080780；3081185-3081373；3089024-3089321；3103043-3103153；3111175-3111310；3115439-3115689；3128184-3128426；3128962-3129092；3130390-3130559；3134161-3134277；3135301-3135443；3141877-3141993；3143654-3143914；3144710-3144820；3147865-3148134；3153144-3153293；3159082-3159352；3162051-3162160；3182821-3183261；3184546-3184823；3191481-3191660；3192387-3192628；3193002-3193115；3193864-3194003；3195486-3196098；3203866-3204440；3206252-3206382；3207299-3207630；3208767-3209099；3213487-3213635；3233906-3234049；3234220-3234322；3265820-3266044；3272171-3272296；3274547-3274736；3276377-3276803；3277493-3277636；3280492-3280771；3282727-3282940；3289767-3290050；3294220-3294333；3302198-3302324；3308428-3308688；3319265-3319553；3321194-3321504；3325631-3325772；3327972-3328117；3331049-3331336；3332089-3332295；3342161-3342279；3351106-3351393；3354296-3354480；3377807-3377935；3378264-3378459；3417006-3417390；3420736-3420941；3421 689-3421949；3425534-3425794；3431290-3431550；3459805-3460107；3460418-3460678；3464723-3464983；3495625-3495795；3504103-3504260；3512718-3512860；3556937-3557273；3669230-3669375；4275026-4275244；4391775-4392024；4736060-4736244；4770676-4770847；5893828-5893942；5917797-5917916；5932351-5932480；6088559-6088667；6111671-6111814；6134181-6134558；6148978-6149125；6159517-6159648；6169924-6170086；6294636-6294911；6341139-6341272；6530699-6530818；6638413-6638578；6664102-6664268；7081270-7081375；7111114-7111237；7723484-7723664；7724019-7724165；7797229-7797491；8086837-8087054；8274043-8274190；8298426-8298685；8403907-8404167；8510416-8510608；10689960-10690091；10917318-10917578；11473186-11473367；11938458-11938578；12251597-12252008；12609996-12610208；13910569-13910698；14156736-14156846；14309883-14310030；15541294-15541554；16061427-16061554；16321032-16321158；16554135-16554558；16861392-16861519；16950647-16950793；17019523-17019823；17026318-17026455；18060459-18060568；18149518-18149705；18686617-18686743；18691983-18692140；19054751-19054854；19665069-19665244；20005721-20005854；21755768-21755917；22927195-22927331；23519947-23520057；26136828-26136971；26503622-26503725；27677607-27677867；27849101-27849266；27994887-27995147；29189324-29189642；30240188-30240341；32169629-32169747；32221936-32222059；32936900-32937089；34420860-34421008；34639624-34639745；36644255-36644374；38181454-38181574；41114654-41114914；41131945-41132084；41253449-41253606；41583010-41583158；41889160-41889394；43392604-43392818；43533970-43534316；43814208-43814324；44031196-44031306；44068789-44068892；45082810-45083346；45271751-45271858；45279449-45279745；46858809-46858941；48452689-48452811；48764353-48764476；53386630-53386996；53528963-53529073；53742195-53742451；53904603-53904799；53935873-53936011；54059896-54059999；54775727-54775970；55521708-55522062；55844167-55844286；56323691-56323793；58521893-58522110；61519888-61520012；62662133-62662237；62783533-62783678；63784342-63784449；68515754-68516070；73571459-73571578；74663494-74663641；74713906-74714135；75590829-75590933；76262373-76262838；82039775-82040055；86577457-86577618；86988863-86989062；90409071-90409331；91189205-91189331；91191796-91192088；94205105-94205238；96135602-96135711；107587472-107587734；110993523-110993625；113291201-113291309；119535588-119535693；119542118-119542296；119549295-119549402；120377669-120377779；144994520-144994637；145092216-145092576；147737221-147737391；149146695-149146855；149148152-149148289；149148886-149149036；149286330-149286476；149857877-149858023；150953712-150953943；151509682-151509791；151810377-151810525；153581750-153581873；153599383-153599573；153934210-153934328；154516404-154516523；155156325-155156430；156047075-156047186；159683404-159683510；159683776-159683891；160178004-160178283；160336399-160336590；160389227-160389339；161947938-161948198；162337162-162337441；166765956-166766137；173579033-173579162；173887148-173887285；179334176-179334289；181098503-181098635；182810641-182810747；194326822-194327068；196547397-196547498；196945529-196945637；197744907-197745026；200005266-200005406；200010631-200010788；200969705-200969906；201475326-201475452；201476557-201476665；202172867-202173002；204531685-204531837；207262319-207262429；207992427-207992724；210857019-210857247；212062720-212063173；214170992-214171132；214638070-214638191；217310965-217311204；218524557-218524685；220552393-220552515；221065070-221065171；222717221-222717387；223567849-223568034；225566690-225566949；225706939-225707215；226651855-226651998；226825434-226825539；227921627-227921778；227947136-227947322；227974825-227975111；228079042-228079222；228289732-228289880；228473852-228474036；228503487-228503829；228528780-228529062；228558892-228559169；230367477-230367737；230513970-230514117；234667362-234667642；235587072-235587249；236228789-236228910；236273193-236273312；238847990-238848099；244080100-244080219；245851484-245851695；246860128-246860388；247542223-247542331；247802774-247803023
10号染色体基因组区域：
415821-415938；461809-462031；577179-577311；579226-579335；664696-664804；665113-665313；1198 688-1198835；1209314-1209498；1228965-1229068；1230849-1230965；1235997-1236180；1245359-1245473；1256493-1256597；1259812-1259956；1260331-1260513；1401818-1402007；1402293-1402426；1506157-1506282；1511206-1511344；1547357-1547625；1583222-1583482；1692977-1693174；1725096-1725230；1789125-1789253；2487078-2487194；3168614-3168777；3172197-3172481；3300397-3300522；3627729-3627972；3724435-3724678；3805226-3805441；3870105-3870305；5580951-5581070；5631674-5631971；5702083-5702221；6919371-6919485；7454580-7454898；8100114-8100286；8100506-8100668；8116869-8117072；10308512-10308678；11923474-11923604；11934141-11934282；19777919-19778061；22158229-22158491；22624674-22624790；23369135-23369236；23480697-23480815；23484032-23484141；24909456-24909607；25010711-25011039；25755061-25755264；30346647-30346789；30720559-30720774；35080974-35081093；35258200-35258474；35928604-35928713；38069487-38069801；42644799-42645192；43247943-43248070；43393702-43393833；43447011-43447171；43697849-43698051；44198410-44198517；45701113-45701247；45799169-45799349；46961780-46961986；49673519-49673645；49954066-49954186；50506754-50507035；54714822-54714950；62023508-62023688；63422511-63422630；65647086-65647213；67189225-67189336；68411485-68411658；71601337-71601482；71892007-71892320；72015285-72015462；72218065-72218443；72727774-72727913；73394970-73395113；73497243-73497523；73521638-73521755；73575412-73575520；73769037-73769219；77794599-77794758；79996996-79997144；81444843-81445113；81904171-81904480；82023899-82024011；82169057-82169163；83068999-83069136；83263584-83263745；86004952-86005070；88703075-88703322；90488114-90488446；91296311-91296510；92592305-92592433；94180416-94180559；94820953-94821086；95123660-95123879；95205504-95205611；98144368-98144497；99160096-99160215；100227560-100227832；101281002-101281262；101282013-101282145；102046685-102046827；102748540-102748734；102778604-102778717；102822249-102822357；102985788-102985915；103530134-103530370；103539228-103539367；103599571-103599692；104535275-104535406；104913266-104913526；105361962-105362081；106011700-106011830；106014437-106014881；106074268-106074603；110672138-110672313；111216768-111216874；118032337-118032898；118434498-118434636；118891118-118891266；120359308-120359441；120543311-120543444；121295914-121296095；122349130-122349232；123371156-123371303；124638868-124638991；125114476-125114594；126256988-126257240；126281463-126281600；126296075-126296434；126711969-126712220；126712372-126712559；129144179-129144307；129535641-129535901；130274349-130274549；130684697-130684873；130855094-130855278；130868340-130868448；130870128-130870256；131354666-131354783；131529354-131529516；131639526-131639751；131706743-131706892；131731385-131731626；132154687-132154824；133204168-133204317；133208394-133208528；133208761-133208879；133644552-133644781；133793645-133793783；133874566-133874833；133921213-133921429；133947644-133947755；133948074-133948227；133949165-133949269；133950676-133950967；133953845-133954068；133961848-133962037；133981439-133981571；133981975-133982102；133983793-133983964；134014432-134014623；134016056-134016336；134016854-134017006；134018801-134019026；134019524-134019727；134021284-134021525；134022557-134022746；134034573-134034936；134040213-134040456；134041500-134041684；134053767-134053915；134055143-134055267；134062149-134062260；134073893-134074009；134085786-134085950；134095204-134095725；134107089-134107228；134117537-134117650；134150489-134150734；134188750-134189010；134219125-134219410；134224956-134225261；134341216-134341361；134495624-134495769；134527948-134528050；134608454-134608563；134610501-134610634；134621538-134621689；134624109-134624244；134647389-134647533；134692498-134692617；134692993-134693128；134694298-134694491；134698676-134698863；134729845-134730203；134732799-134733122；134735972-134736112；134738977-134739370；134740633-134740899；134790766-134790938；134791519-134791769；134793947-134794059；134806480-134806584；134837866-134838078；134838342-134838596；134842578-134842818；134844342-134844924；134861023-134861180；134875725-134875916；134881949-134882099；134884125-134884280；134894796-134895108；134896348-134896638；134908186-134908325；134913507-134913620；134913890-13491 4022；134928095-134928246；134928613-134928749；134938102-134938249；134945401-134945727；134973001-134973107；134979125-134979261；134980505-134980628；134980789-134981053；134981669-134982010；134993121-134993323；134996378-134996494；134998356-134998578；135001547-135001673；135005324-135005461；135016400-135016541；135022993-135023127；135026828-135026943；135032460-135032633；135105810-135106070
11号染色体基因组区域：
433315-433423；460626-461036；491225-491374；562173-562629；563859-564043；569264-569546；598324-598690；800391-800661；840419-840522；841236-841377；1009590-1009740；1025794-1026026；1027555-1027730；1077546-1077671；1213283-1213534；1215670-1215847；1216193-1216453；1238618-1238853；1252151-1252272；1258450-1258590；1263257-1263411；1263729-1264000；1264798-1265058；1275970-1276123；1326436-1326546；1331584-1331785；1464311-1464480；1471563-1471675；1661630-1661737；1824048-1824490；1892042-1892375；1918275-1918482；1940911-1941171；1948916-1949221；1976431-1976556；1977556-1977702；1983001-1983194；2016351-2016626；2150583-2150730；2152311-2152513；2154047-2154952；2156307-2156544；2308361-2308488；2573990-2574106；2594839-2594942；2610068-2610195；2828647-2828779；2924858-2925027；2942754-2942867；5290745-5290849；6340684-6340944；8378686-8378973；11832933-11833213；15136178-15136295；15963013-15963197；17591774-17591902；17742018-17742147；19735182-19735320；19736191-19736471；22454151-22454499；23286497-23286693；23421709-23421953；23427917-23428068；23674871-23675037；23905153-23905261；27672696-27672807；32450424-32450591；35440031-35440158；35808666-35808786；35966361-35966568；39462691-39462987；41644388-41644520；44087634-44087748；44087973-44088080；44541358-44541464；45827403-45827875；46401359-46401504；46709238-46709341；47208979-47209160；47613367-47613484；47927067-47927327；56099764-56099898；57232348-57232508；58912211-58912327；59667014-59667119；60383157-60383407；61096982-61097114；61687817-61687937；61900808-61900991；61957678-61957809；63783225-63783416；63784025-63784166；64064979-64065125；64127709-64127816；64140341-64140470；64374712-64374819；64863168-64863379；64992997-64993107；65313934-65314043；65314375-65314541；65321406-65321662；65359412-65359570；65360017-65360227；65374749-65375224；65487450-65487551；65582798-65582918；65768425-65768673；66193938-66194128；66458798-66459081；66472290-66472413；66838979-66839093；67210269-67210384；67278778-67278904；67494341-67494473；68133047-68133219；68135679-68135932；68138818-68139042；68860016-68860122；69259196-69259335；69813138-69813530；70741247-70741377；71623045-71623320；72533239-72533357；74178113-74178268；74440515-74440645；76025109-76025321；76849076-76849217；80242887-80243022；80918570-80918873；81662731-81662889；82114950-82115250；85194996-85195305；85646499-85646618；88241585-88241766；89522850-89522959；92600068-92600334；93475896-93475999；94883424-94883564；112505587-112505690；113930736-113931017；114112901-114113129；114479593-114479698；114484683-114484831；116228521-116228700；117069780-117070018；117314862-117314964；117376078-117376338；117541468-117541600；119181451-119181587；119317202-119317399；119473914-119474019；119524039-119524211；120384015-120384150；121349643-121349767；123016009-123016126；124953019-124953193；126985893-126986034；127811623-127811817；128778360-128778620；129613524-129613831；129731669-129731883；129734455-129734599；130060344-130060521；131130899-131131039；131561146-131561268；131939119-131939245；131939970-131940174；132016219-132016419；133800709-133800949；134253340-134253625；134253820-134253954；134393379-134393576；134794919-134795051
12号染色体基因组区域：
296473-296579；562849-563109；718054-718314；1059330-1059457；1937782-1937899；1948379-1948579；1973812-1973944；2215413-2215574；2787762-2787912；2788752-2788900；2800985-2801117；3309751-3309886；3310077-3310303；3529673-3529815；4543223-4543336；4554886-4555027；6729405-6729553；6745460-6745569；6946126-6946231；6959518-6959658；7062109-7062240；7071780-7071921；7167780-7167900； 7583650-7583756；7781092-7781289；9892249-9892632；10251413-10251678；10264269-10264384；11760584-11760854；12419035-12419209；12618535-12618660；15654718-15654837；15743119-15743233；20094183-20094296；20267333-20267475；20268256-20268405；20705364-20705772；20935613-20935748；23439187-23439314；27924231-27924397；31270307-31270456；34471039-34471144；34475337-34475448；34478217-34478389；34489902-34490032；34491014-34491157；34516115-34516471；41086179-41086327；41966249-41966400；42979452-42979569；48921680-48922002；51717595-51717866；51718108-51718504；52258077-52258299；52309016-52309296；52408677-52408937；52540565-52540825；52636850-52637020；52789509-52789749；52994767-52995027；53453100-53453221；54408027-54408250；54422347-54422473；54441169-54441496；54447632-54448038；56031025-56031143；57290257-57290375；57351092-57351229；57569768-57569899；57599348-57599457；58000654-58000881；58013414-58013620；59689560-59689723；65218216-65218372；67072768-67073063；69198710-69199108；74564430-74564636；74564803-74564915；74796732-74796882；85187890-85188120；94533095-94533250；94580419-94580658；94676506-94676709；94939915-94940020；100041843-100041975；100823976-100824406；103696137-103696285；108985716-108985817；110270879-110271025；110506167-110506299；114236455-114236567；114263695-114263833；114877844-114877979；115131420-115131546；116354803-116354947；116808768-116809048；117579965-117580102；117627099-117627223；121343016-121343149；121661747-121661868；121890488-121890605；122356821-122356952；123215107-123215239；123738214-123738340；124393810-124394029；124773720-124773980；124870076-124870515；125217602-125217729；125271270-125271435；125627229-125627489；127650559-127650674；127965770-127965882；128869789-128870093；129148451-129148615；129190447-129190574；129468126-129468250；130184739-130184851；130384863-130385014；130572620-130572734；130683456-130683640；130840928-130841078；130908854-130909059；130909641-130909756；131194033-131194140；131366591-131366877；131367209-131367428；131437900-131438178；131529236-131529419；131572549-131572674；131605592-131605749；131850468-131850600；131865276-131865380；132169193-132169459；132280441-132280706；132620803-132621063；132663422-132663562；132680733-132680973；132682573-132682807；132687626-132687777；132688219-132688359；132688933-132689064；132822420-132822665；132824316-132824425；132837350-132837796；132839020-132839169；132848607-132848729；132849401-132849545；132851967-132852087；132863714-132864221；132880775-132881162；132895296-132895543；132898924-132899096；132952634-132952755；132956419-132956787；132980605-132980728；132981736-132981926；132982275-132982565；132987039-132987176；133004858-133004971；133012177-133012599；133056699-133056977；133100669-133100773；133101094-133101225；133120445-133120848；133123407-133123624；133135487-133135686；133159420-133159843；133160460-133160573；133161622-133162062；133177725-133177883；133197770-133198064；133336998-133337158
13号染色体基因组区域：
19582812-19582925；20392317-20392577；20980095-20980206；21291152-21291297；22615324-22615469；22637826-22637943；23458123-23458364；25801840-25802080；25946831-25947200；29107238-29107373；29148868-29148980；29192850-29193054；29933449-29933584；30054342-30054522；30071293-30071553；30077289-30077510；36788555-36788815；37453407-37453512；43134712-43134866；47014718-47014820；49793020-49793280；49921802-49921924；51568062-51568207；51825835-51826017；52981271-52981395；53422319-53422461；53422649-53422791；54025812-54026149；60015420-60015540；64314939-64315074；70681866-70681992；72249231-72249343；73636077-73636209；76279069-76279346；79982788-79982944；81064032-81064280；84061235-84061374；91148199-91148442；95355521-95355646；97599730-97600054；99095683-99095796；99096364-99096516；99096713-99096865；99383487-99383747；99629851-99630223；99737372-99737705；100621635-100621800；101315183-101315464；109616321-109616435；110533158-110533302；110781474-110781749；110965644-110965914；111971757-111971961；111972733-111973057；112111066-112111209；112200412-112200534；112200745-112201013；112225448-112225606；112238555-1122388 73；112255469-112255588；112507644-112507788；112517379-112517505；112555972-112556085；112712023-112712137；112817732-112817855；112996304-112996422；113291762-113292022；113409882-113409987；113424098-113424250；113464307-113464413；113544390-113544674；113552480-113552740；113553780-113553891；113585777-113585948；113613573-113613717；113634165-113634459；113648761-113649031；113670884-113671297；113678805-113679064；113689097-113689377；113700520-113700641；113701833-113702105；113708541-113708801；113714316-113714454；113718282-113718430；113719005-113719285；113719915-113720039；113731198-113731511；113735268-113735374；113739025-113739426；113741556-113741663；113742704-113742989；113752386-113752786；113752892-113753008；113795921-113796043；113803178-113803397；113811492-113811630；113991973-113992129；114074346-114074466；114074681-114074839；114107876-114108141；114185609-114185752；114186002-114186117；114187890-114188150；114311777-114312053；114312456-114312743；114579117-114579288；114748219-114748723；114761897-114762157；114766724-114767088；114768170-114768522；114770057-114770194；114774490-114774623；114774937-114775106；114776333-114776480；114782674-114782839；114801076-114801190；114801472-114801632；114802547-114802648；114807120-114807306；114828289-114828436；114846514-114846754；114862690-114863026；114872714-114872887；114875170-114875467；114917465-114917735
14号染色体基因组区域：
19552756-19552882；19888527-19888729；21469383-21469528；22370643-22370777；24546392-24546540；24779925-24780064；24780464-24780825；29228334-29228475；29254667-29254796；35871714-35871957；36987167-36987432；37127671-37127775；38067839-38067989；42075553-42075695；47289472-47289732；47669846-47669972；48095758-48095872；50789276-50789536；53684197-53684562；54860350-54860477；57833188-57833298；58864739-58864887；59065986-59066095；59112362-59112725；59261405-59261517；61103979-61104118；61110728-61110851；65289804-65289908；68038823-68038973；69256594-69256946；70039176-70039871；70316866-70317048；71634121-71634340；71635170-71635289；71720841-71721359；72757827-72757933；72976906-72977078；73396377-73396516；73706424-73706539；77730976-77731087；78709726-78709906；81790233-81790391；82089218-82089329；82145655-82145878；90162180-90162322；90967496-90967644；91880085-91880203；93154317-93154424；93170710-93170831；94173126-94173261；94306818-94306932；94393652-94393911；94417396-94417505；94776104-94776285；95358041-95358143；95942665-95942828；97431262-97431368；99665186-99665321；101198727-101198831；101235838-101235964；101290704-101290986；101293029-101293167；101341254-101341377；101347193-101347429；101498224-101498354；101506232-101506440；101509249-101509363；101512542-101512896；101515918-101516066；101618124-101618439；101967708-101967920；102027719-102028123；102053948-102054054；102101574-102101696；102974004-102974264；103294573-103294770；103411329-103411460；104151982-104152124；104600454-104600634；104622058-104622185；104638075-104638634；104717199-104717317；104741838-104742148；104800629-104800730；105012456-105012634；105103000-105103123；105154789-105154975；105156970-105157120；105196085-105196345；105196441-105196564；105218119-105218379；105220620-105220774；105247214-105247320；105282932-105283053；105318278-105318380；105332541-105332644；105538413-105538545；105715648-105716016；105741936-105742116；105858433-105858573；105930383-105930533；105936287-105936429；105964494-105964647；106236068-106236301；106744195-106744492
15号染色体基因组区域：
20500097-20500216；21260243-21260383；22954676-22954817；25328405-25328598；25331538-25331810；25333245-25333481；25333693-25333819；25429120-25429262；25439336-25439494；25452087-25452234；25454353-25454613；25468463-25468600；25475546-25475655；25961763-25962039；26107480-26107780；27494330-27494511；27786834-27786990；28197404-28197546；28272391-28272548；28834235-28834341；29210460-29210609；29210940-29211091；31295015-31295133；31685126-31685238；32933783-32933978；35591628-35591775；40544352-40544515；41165190-41165335；42371635-42371842；45670999-45671279；4 6501861-46501985；53090486-53090611；53095771-53096242；54754511-54754719；55881585-55881853；60287445-60287663；61517509-61517629；62516463-62516607；63312170-63312301；63671591-63671693；65503571-65503848；66274691-66274862；67073455-67073597；67418251-67418396；68096933-68097080；68549114-68549256；68624774-68624886；70168931-70169146；72095117-72095298；72490399-72490637；72524585-72524688；74709660-74709781；75019250-75019393；75111015-75111157；75136035-75136181；76408348-76408488；76629837-76629951；77272025-77272146；77320669-77320797；77324526-77324734；77861577-77861688；77928918-77929063；77933713-77933918；78196323-78196536；79043911-79044028；79471437-79471561；81410799-81410916；81426635-81426854；81666392-81666512；86126117-86126407；89198776-89198878；89876688-89877101；89954947-89955060；90320166-90320508；90578559-90578665；91840099-91840252；92706010-92706210；95399900-95400006；96906421-96906807；98647804-98647959；99966831-99967113；100672241-100672497；101169888-101170019；101626657-101626849；101695660-101695761；102520336-102520448
16号染色体基因组区域：
351770-351955；476939-477199；507982-508180；560102-560280；585815-585961；605144-605282；646387-646514；672266-672502；696429-696551；727196-727298；788079-788184；798264-798440；810594-810696；811028-811141；816004-816228；818329-818501；832991-833213；874313-874422；874629-874894；883878-883999；891200-891402；893730-894068；894780-895048；895260-895474；900744-900944；921005-921187；922023-922174；926158-926321；928931-929102；938037-938142；941822-942231；945521-945676；959675-959816；960017-960186；963281-963404；967900-968132；968136-968396；970628-970888；1001256-1001363；1017696-1017868；1022357-1022462；1024260-1024381；1027310-1027510；1033335-1033485；1034330-1034448；1040572-1040708；1056983-1057140；1069552-1069816；1071036-1071349；1076847-1076989；1077614-1077762；1112280-1112395；1116317-1116428；1128048-1128184；1129936-1130062；1131907-1132125；1138264-1138405；1138697-1138798；1146160-1146337；1151097-1151288；1158502-1158617；1179659-1179845；1204875-1205026；1207890-1208006；1210774-1210887；1215465-1215641；1216737-1216876；1217148-1217292；1217827-1217971；1221814-1222232；1236204-1236522；1244320-1244467；1256258-1256367；1257708-1257906；1261243-1261399；1262087-1262230；1271879-1271985；1273762-1273911；1275480-1275582；1305586-1305813；1365801-1365915；1369206-1369335；1394962-1395111；1397728-1397878；1404850-1405063；1487459-1487703；1493756-1493860；1500343-1500507；1560800-1561275；1570743-1571003；1585920-1586076；1795200-1795552；1796810-1797449；1813975-1814251；1843625-1843830；2012706-2012914；2040930-2041062；2140870-2141007；2203175-2203316；2273017-2273159；2288601-2288702；2338129-2338282；2358275-2358417；3059648-3059836；3493359-3493504；3706674-3706785；3989432-3989550；4714033-4714152；4730391-4730767；4732926-4733257；6069932-6070116；6533180-6533460；7382335-7382477；7796774-7796925；10935153-10935344；14397504-14397942；14400873-14401025；19126144-19126395；19127248-19127359；21358305-21358443；22326413-22326543；25159910-25160172；27234298-27234558；27853395-27853526；28081140-28081267；28224136-28224249；28331351-28331466；28948278-28948409；29118820-29118983；29151048-29151200；29151523-29151674；29169963-29170230；29222347-29222448；29262792-29263025；29323906-29324009；29340601-29340743；30017955-30018195；30100355-30100511；30124686-30124904；30133122-30133311；31146861-31147128；31483162-31483317；32290058-32290182；33070350-33070866；33509682-33509903；33852490-33852800；34208387-34208655；35027326-35027548；35029898-35030009；46962905-46963079；46963989-46964158；49525631-49525806；49669731-49669842；49686461-49686668；49783345-49783635；50745959-50746088；50873851-50873953；51026677-51026962；51184280-51184462；54318157-54318314；56659375-56659635；56709801-56710036；57669429-57669569；57831641-57831859；57832134-57832309；57836787-57836913；58120934-58121038；58131326-58131428；58191975-58192104；63163378-63163649；65105709-65105857；65157743-65158003；66918914-66919049；66943845-66943952；67183642-67183774；67918699-67918926；68014177-68014303；68269362-68269515；69776038-69776369；7 0812830-70813090；71571085-71571203；73100458-73100568；75148407-75148550；75263716-75263905；75283549-75283661；75512693-75513169；75550021-75550165；77465154-77465272；79027500-79027745；80654677-80654787；81526928-81527067；81527249-81527524；81533151-81533275；81536133-81536289；81564093-81564528；81565706-81565886；81667103-81667207；82661521-82661623；84417988-84418092；84483628-84483766；84492560-84493006；84722861-84722990；85075042-85075233；85196346-85196615；85209162-85209325；85256212-85256366；85337322-85337537；85363003-85363117；85458281-85458415；85608003-85608311；85650374-85650557；85678088-85678415；85966415-85966519；85969300-85969411；86160817-86160950；86184977-86185116；86528271-86528385；86714536-86714745；86715028-86715325；86772057-86772158；86774040-86774154；86795398-86795511；86911938-86912087；86951582-86951715；87451112-87451214；87641064-87641189；87682628-87682743；87740820-87740930；87864445-87864567；87867921-87868087；87904158-87904547；88052922-88053108；88315446-88315572；88330720-88330954；88371318-88371488；88454810-88454952；88476959-88477074；88480700-88480885；88482183-88482352；88493595-88493702；88504983-88505222；88744579-88744891；88821809-88821926；88832470-88832814；88884969-88885095；88905033-88905257；88905408-88905745；88941486-88942093；88945738-88945937；88946849-88947069；88963090-88963306；88964439-88964944；89000094-89000311；89000900-89001002；89009874-89009998；89019476-89019670；89050730-89050833；89100795-89100933；89115674-89115834；89120615-89120840；89137796-89137959；89141880-89142023；89229268-89229369；89346234-89346386；89387014-89387519；89461615-89461907；89640889-89641060；89688060-89688504；89900088-89900250；89900350-89900765；89972422-89972926；89973141-89973267；90128717-90128834
17号染色体基因组区域：
86514-86774；146378-146508；263405-263591；707114-707265；708439-708583；709220-709449；710617-710890；730169-730313；744589-744849；747395-747596；750777-750960；754213-754329；805425-805647；822721-823014；950471-950649；951755-951962；1029686-1029866；1093963-1094351；1395854-1395960；1474682-1474879；1494318-1494471；1553414-1553592；1634125-1634566；1881105-1881214；1961565-1961701；3716200-3716611；4510577-4510683；4803421-4803764；4979072-4979184；5019385-5019496；7727101-7727219；7754799-7755013；7792052-7792191；7836524-7836640；8110065-8110351；8791859-8791988；9976391-9976504；10212329-10212602；10422077-10422190；14207369-14207512；16849251-16849389；17062034-17062170；17088577-17088684；17110182-17110353；17359835-17359973；17407805-17407909；17713349-17713719；17910196-17910434；18088312-18088578；19220794-19220904；19350000-19350102；19617148-19617305；21731120-21731268；22020651-22020804；22253212-22253324；25335912-25336049；25798733-25799019；25993431-25993537；27044064-27044320；27893094-27893331；28562117-28562218；30348178-30348317；30348728-30348898；30367336-30367458；32366789-32367099；33672833-33672973；34097374-34097546；36997619-36997797；37042200-37042329；37074968-37075238；37331612-37331923；38024208-38024476；39680318-39680724；40274686-40274946；40275266-40275382；40308110-40308252；40318355-40318543；40330818-40330951；40713882-40714255；40715043-40715407；40805862-40805984；40824078-40824379；41847096-41847236；42431865-42432006；42635625-42636182；43198355-43198493；43862893-43863023；44090853-44090999；46138081-46138182；46227996-46228261；46607782-46608135；46673394-46673560；46799639-46799745；46804207-46804415；47113489-47113597；48628400-48628516；48653108-48653296；49746979-49747091；55474061-55474183；55533020-55533208；56621341-56621578；59534544-59534683；60729461-60729588；61995743-61995860；62019271-62019377；62066668-62066826；63225002-63225127；65052129-65052231；66375071-66375196；70113454-70113585；70637038-70637333；72345389-72345493；73088154-73088350；73126515-73126665；73696459-73696655；73719830-73720014；73720590-73720840；73739801-73739941；73754448-73754584；73805841-73806157；73914273-73914375；73996169-73996384；74005350-74005578；74133474-74133786；74222881-74223128；74908575-74908882；75385014-75385117；75385329-75385458；75522239-75522375；75558356-75558632；75797020-75797129；76183640-76183779；7641789 3-76418003；76422911-76423049；76425170-76425309；76425465-76425595；76472296-76472590；76967614-76967826；77030191-77030327；77111616-77111727；77224269-77224403；77393894-77394062；77419496-77419669；77460269-77460399；77680318-77680444；77685550-77685707；77808309-77808503；77817346-77817523；77834120-77834281；77884831-77885163；77924314-77924422；77948976-77949105；78064033-78064293；78090499-78090759；78293360-78293470；78417889-78418088；78444608-78444747；78748439-78748552；78937786-78937961；78939669-78939807；78940033-78940172；78999635-78999909；79017352-79017536；79030900-79031091；79045455-79045664；79096477-79096676；79099777-79100287；79108111-79108257；79228880-79229024；79388434-79388754；79430811-79431136；79432888-79433066；79477840-79478078；79494897-79495544；79495659-79495929；79514682-79514849；79680645-79680803；79827105-79827226；79827601-79827913；79936502-79936625；79957456-79957563；79961397-79961639；79970070-79970310；80022818-80023034；80038280-80038385；80278862-80278971；80290004-80290278；80337468-80337614；80350283-80350408；80351067-80351349；80393104-80393216；80615580-80615693；80621636-80621770；80654550-80654855；80794148-80794322；80798013-80798130；80834070-80834238；80841388-80841593；80845456-80845654；80867522-80867718；81014671-81014811；81042475-81042693；81042957-81043190
18号染色体基因组区域：
2913099-2913359；4692168-4692315；6929695-6929973；7011217-7011517；11147324-11147438；11148502-11148704；12093605-12093787；12254608-12254799；12376166-12376287；12911025-12911263；19997912-19998018；30253077-30253179；32557856-32557978；35142911-35143018；36523511-36523656；36524696-36524815；41901006-41901266；44260325-44260450；44777838-44777945；54814561-54814670；55018649-55018791；55019621-55019881；55102008-55102159；59483326-59483510；61143964-61144293；63673366-63673522；67918103-67918363；68098182-68098309；74062554-74062691；74091419-74091536；74091849-74091962；74173808-74173967；75691288-75691435；75998530-75998790；76601754-76601899；76690786-76690906；76764164-76764328；77181332-77181458；77211083-77211208；77218368-77218521；77233391-77233493；77272517-77272669；77289042-77289214；77313759-77314027；77612132-77612280；77622411-77622671；77623511-77623889；77638029-77638277
19号染色体基因组区域：
407274-407401；426996-427264；440739-441012；641771-641985；643216-643390；736374-736475；751124-751242；812514-812639；863075-863226；929476-929834；939522-939782；944234-944353；1008998-1009312；1009581-1009978；1012076-1012181；1105470-1105989；1112618-1112890；1163467-1163571；1207406-1207645；1220659-1220815；1307442-1307702；1308088-1308193；1409489-1409611；1440259-1440375；1465904-1466033；1496187-1496464；1785443-1785546；1800116-1800276；1881012-1881272；1985881-1986159；1993188-1993317；2038426-2038539；2275664-2275933；2276231-2276566；2291272-2291834；2356433-2356558；2358620-2358743；2430116-2430394；2511569-2511714；2700642-2700988；2717355-2717493；2980113-2980244；3344155-3344281；3481765-3482028；3507795-3507932；3539061-3539203；3669758-3669894；3905467-3905577；3964708-3964989；3984172-3984323；4091049-4091217；4391326-4391534；4552455-4552575；4556028-4556131；4792724-4792836；5210577-5210692；5240143-5240245；5244109-5244234；5250308-5250508；5335108-5335261；5691805-5692212；5822190-5822490；5829147-5829298；6007848-6007957；6534759-6534917；7267899-7268027；7293222-7293352；7505139-7505453；7626084-7626198；7712038-7712372；7926980-7927286；7928046-7928163；8387233-8387343；8398613-8398727；8654344-8654451；8676733-8676862；9370151-9370331；9965432-9965583；10076989-10077270；10254525-10254637；12306037-12306438；12306467-12306581；12758957-12759157；12880553-12880844；12969320-12969630；13110697-13110946；13198813-13198999；13365937-13366152；14359679-14359987；15281126-15281608；15487839-15487991；15564032-15564292；15719695-15719824；16198791-16198996；16199301-16199472；16528768-16529028；17283537-17283942；17877723-17877846；17941985-17942360；18545021-18545140；18557105-18557380；18572345-18572546；18888694-18888799；18979170-18979504；19648029-19648456；22123507-22123704；2219 3425-22193710；22682209-22682341；24182604-24182719；30155520-30155714；31770037-31770174；33697975-33698181；34263975-34264112；35715867-35715996；35764609-35764806；35800808-35801031；36024802-36024954；36365483-36365713；36435460-36436056；36499325-36499477；36987989-36988110；38284033-38284143；38307971-38308263；39009920-39010101；39051851-39052224；39217542-39217681；39921424-39921539；40676400-40676563；40711801-40711929；41313752-41313856；41732534-41732669；41836965-41837171；42703790-42703900；42906601-42906740；43912178-43912594；45211154-45211314；46032452-46032732；46056784-46057103；46806880-46807331；48228952-48229220；48244318-48244419；48725593-48725714；49059650-49059790；49116355-49116496；49120443-49120595；49238619-49238762；49517745-49517860；49971643-49971789；49993157-49993268；50027789-50027925；50364420-50364533；50365147-50365360；51021468-51021728；51132585-51132706；51174888-51175001；51535251-51535466；51917729-51917918；52104320-52104624；52216745-52217059；56090047-56090307；56603156-56603394；58549185-58549455；58571667-58571816；58629729-58629901；58958222-58958364
2号染色体基因组区域：
314941-315145；502994-503242；503471-503857；621816-621968；816675-816823；817583-817715；866740-866878；882039-882307；883681-883793；906893-907022；1017930-1018077；1130469-1130642；1151708-1151813；1166225-1166333；1237456-1237607；1291358-1291471；1480763-1480992；1482854-1483028；1497746-1497869；1543374-1543485；1566909-1567411；1792515-1792762；1846718-1846845；1895732-1896006；1941126-1941290；1997887-1998264；2831009-2831267；2875964-2876079；3151358-3151466；3185199-3185469；3452412-3452672；3749733-3749993；3818767-3818868；3834250-3834353；5896451-5896566；8595989-8596120；8714184-8714530；9347890-9348018；9375569-9375768；9533797-9533907；10151636-10151908；10152738-10152914；10153821-10153967；10154227-10154365；10384631-10384732；10567046-10567251；10619227-10619346；11076220-11076327；11750747-11751009；11758654-11758793；11925414-11925532；12880419-12880684；15703422-15703623；16154211-16154334；22059106-22059217；23913308-23913569；23913772-23913932；24397744-24398076；25051006-25051131；25475724-25475928；27529746-27529898；27530769-27531598；27665507-27665639；27958421-27958530；30144168-30144384；31215668-31215914；31359819-31359936；31457607-31457722；33953223-33953352；42319246-42319372；45168776-45169036；45169422-45169774；47419168-47419278；59182247-59182386；59477247-59477383；62116650-62116822；63280428-63280532；63285364-63285519；63286469-63286709；64834105-64834431；64876959-64877091；68251455-68251561；68675722-68675940；71134002-71134122；71205519-71205998；71211845-71212013；71294876-71295004；73161953-73162159；73164590-73164723；73496066-73496180；74347459-74347734；74668353-74668477；76672413-76672525；78182097-78182272；80101281-80101401；83164674-83164776；83212913-83213047；84105399-84105599；86307198-86307394；88124901-88125161；89064507-89064654；96097860-96097961；97174770-97174934；97652205-97652349；99013246-99013410；99439906-99440028；102091153-102091362；105697669-105697796；107459775-107459882；109559228-109559453；109934688-109934960；113379619-113379780；114048867-114049009；114325955-114326073；117813669-117813782；118617169-118617429；119591601-119591704；119606694-119606799；120190151-120190368；121116537-121116640；122088551-122088676；124441553-124441710；124445363-124445503；125745049-125745172；126595222-126595338；127801049-127801356；127963474-127963591；128389096-128389237；129131469-129131617；129661444-129661593；130691090-130691215；131520256-131520421；132220155-132220415；132724040-132724161；133426718-133426907；134024317-134024467；135568278-135568379；140167808-140167942；142022623-142022728；147344967-147345072；147345256-147345399；148444157-148444333；148584353-148584816；159726425-159726561；161127322-161127688；162275374-162275596；164204508-164204841；164960000-164960125；165874788-165874926；168572451-168572698；171679000-171679139；172430711-172430816；174890808-174890957；175191840-175192170；175412124-175412755；175595399-175595599；176932629-176932886；176964588-176964835；178972945-178973081；192745999-192746104；192746300-1927464 34；194367473-194367574；198571482-198571682；200213525-200213630；200331902-200332063；200334900-200335072；202004720-202004884；202737401-202737676；207305207-207305318；216274620-216274747；217674547-217674756；218843925-218844078；218988817-218988956；219128422-219128551；219219509-219219647；219256054-219256164；219735751-219736010；219818548-219818739；219857167-219857345；220406607-220406771；223170537-223170797；226608712-226608855；228185299-228185452；232546136-232546246；233245980-233246089；233251745-233251882；233385141-233385262；233757562-233757690；235372806-235372935；236415626-236415844；236773979-236774083；236777077-236777194；237068534-237068794；237964992-237965104；238280909-238281017；238289880-238290023；238446575-238446715；238617110-238617278；238621262-238621443；239048106-239048361；239048462-239048722；239317900-239318175；239495105-239495227；239709312-239709428；239865187-239865288；239949254-239949449；239951168-239951316；240002094-240002278；240005916-240006019；240029685-240029806；240039954-240040096；240100625-240100822；240168211-240168360；240449613-240449764；240738007-240738359；240748209-240748358；241076094-241076361；241098608-241098728；241185018-241185290；241197081-241197297；241389046-241389281；241390051-241390223；241391057-241391185；241393303-241393414；241440038-241440250；241568341-241568630；241586162-241586450；241613203-241613319；241807708-241807827；241835226-241835462；241835670-241835799；241851342-241851539；241896876-241897033；241988020-241988125；242004860-242005162；242011111-242011216；242212500-242212617；242294868-242295000；242509492-242509709；242549382-242549530；242598716-242598840；242683938-242684066；242754587-242754771；242787213-242787398；242794882-242795200；242813983-242814188；242815135-242815331；242833054-242833255；242833458-242833634；242939254-242939481；242988004-242988116
20号染色体基因组区域：
824493-824665；3218090-3218216；3766241-3766611；5451914-5452066；6194166-6194328；13279934-13280297；17557017-17557143；17595272-17595449；18024141-18024285；18489655-18489766；22559740-22559924；29955962-29956063；30195968-30196163；30777591-30777894；30778219-30778329；31041369-31041476；32379831-32380033；34638218-34638423；34699991-34700251；34995363-34995513；36148604-36148799；36151119-36151269；36153186-36153309；37352085-37352229；42788668-42789043；43378708-43378895；43926929-43927189；44839110-44839279；48598339-48598445；48768685-48768804；57408794-57408913；57582971-57583196；58514042-58514316；59965709-59965816；59969316-59969456；60441927-60442057；60448702-60448813；60501966-60502086；60520205-60520306；60639273-60639496；60953364-60953667；60971545-60971715；61041540-61041645；61151636-61152007；61153871-61153998；61158286-61158387；61314855-61314986；61318364-61318594；61403308-61403478；61905169-61905384；61923786-61923900；61939628-61939773；61978048-61978194；61979533-61979667；61986016-61986161；62002864-62003022；62031707-62031925；62032309-62032469；62032823-62032984；62037630-62037758；62084647-62084825；62097753-62097994；62111155-62111423；62126196-62126299；62127292-62127395；62679364-62679725；62729774-62730203；62813809-62813966；62818658-62818811
21号染色体基因组区域：
10596670-10596813；10597540-10597841；10599393-10599655；10991067-10991339；10994948-10995059；28515757-28515863；28516095-28516200；30372612-30372752；30451257-30451380；33247238-33247417；33805624-33805734；34166477-34166641；37618727-37618869；37667907-37668053；37852547-37852666；38066839-38066972；39493180-39493503；39643732-39643869；43185715-43185832；44106225-44106545；44352528-44352698；44369619-44369813；44389866-44389987；44473691-44473840；44480585-44480711；44721027-44721167；44821312-44821560；45582426-45582529；45622342-45622614；45622803-45623168；45664074-45664211；45683165-45683429；45705485-45705980；45709420-45709742；45712762-45713022；45713606-45713720；45752382-45752598；45881450-45881710；45926462-45926643；46321493-46321761；46348236-46348364；46410851-46410953；46421181-46421290；46424706-46424944；46521035-46521228；4681 6568-46816698；46850377-46850665；46859124-46859262；46918409-46918557；46929780-46930079；46935441-46935701；46964194-46964343；46972437-46972588；46972744-46972914；46973298-46973462；47286962-47287426；47288359-47288844；47292177-47292324；47318755-47318961；47322452-47322554；47394303-47394563；47404222-47404424；47406763-47406966；47421037-47421389；47422541-47422683；47423533-47423693；47453296-47453556；47533224-47533354；47545624-47545761；47551773-47552605；47560278-47560438；47560678-47560830
22号染色体基因组区域：
16123154-16123315；16228363-16228507；17518180-17518440；17897894-17898235；18260220-18260480；18639239-18639444；19165207-19165314；19710954-19711270；19892663-19892769；19960370-19961052；20133575-20133687；20134349-20134530；20716156-20716416；20783863-20783966；20784984-20785169；22007099-22007238；22058008-22058268；23523866-23524229；23776101-23776234；23794252-23794534；23799716-23799835；23802510-23802761；24115526-24115685；24224867-24225017；25575338-25575485；25761780-25761992；26148670-26148940；28007090-28007375；28193815-28194075；28194970-28195230；29445638-29445746；29706708-29706823；30115202-30115349；30125363-30125561；32014284-32014544；34046406-34046577；36091773-36091963；37420264-37420524；37493705-37494160；37499342-37499452；37499607-37499742；37608625-37608846；37678641-37678791；37771204-37771443；38484049-38484176；38506545-38506781；39712955-39713087；39770340-39770557；40019115-40019304；40058029-40058146；40814826-40815387；41185198-41185342；42523474-42523617；44708907-44709095；45018513-45018654；45132873-45133362；46508320-46508608；46770059-46770319；46774141-46774289；46787377-46787562；46859899-46860049；46929628-46929829；47513273-47513533；48977330-48977449；49138260-49138539；49141691-49141847；49295437-49295575；49376409-49376857；49579808-49579999；49717597-49717905；49761875-49762158；49766838-49767010；49795411-49795573；49812104-49812350；49843537-49843801；50158120-50158267；50219681-50219943；50470104-50470482；50479987-50480130；50720723-50720864；51043305-51043407；51043672-51043785；51143196-51143299
3号染色体基因组区域：
9969911-9970024；10491154-10491268；12829922-12830031；12856794-12856903；13028501-13028781；13555611-13555722；13694350-13694484；14853147-14853268；16119166-16119269；16925454-16925569；27353058-27353176；27754351-27754494；38175455-38175600；42056643-42056787；42265624-42265750；48310428-48310599；48677634-48677768；49394621-49394731；49394890-49395074；49689805-49690101；49824444-49824559；49947831-49947935；51746670-51746989；51749763-51749960；52099372-52099549；52478749-52478853；53033025-53033341；57543312-57543472；57743413-57743704；62358080-62358370；70908960-70909102；75263610-75263718；77089354-77089475；77289659-77289795；83266692-83266798；85008633-85008780；87424596-87424853；93637424-93637531；96393715-96393862；99594791-99595300；101406231-101406491；103447185-103447336；109115208-109115441；112706474-112706601；112998694-112998819；114647005-114647115；114903881-114904056；120170436-120170540；121903514-121903626；125677240-125677349；126679198-126679343；128210766-128211158；128211495-128211625；128212214-128212337；128336772-128336889；128998518-128998630；129866878-129866992；134635235-134635336；138170578-138170802；140866487-140866669；149768332-149768452；149768479-149768601；153096365-153096626；156837968-156838228；157821357-157821617；159754100-159754204；161214690-161214923；168416772-168416921；169470001-169470137；169539942-169540210；169540298-169540688；170303580-170303706；170626721-170626841；172858838-172859108；180042402-180042574；181421427-181421570；183209625-183209915；183952495-183952633；184280643-184280825；190105485-190105745；193272561-193272870；193857480-193857624；194014564-194014723；195489708-195489980；195587085-195587234；195849415-195849751；195869881-195870141；196326326-196326457；196522757-196522934；197184326-197184440；197238913-197239159；197388168-197388327；197401717-197401995
4号染色体基因组区域：
52892-53692；298812-298963；565894-566007；619766-619869；629011-629152；631732-631867；660260-660466；681236-681347；686969-687078；786763-786883；793002-793262；798555-798662；812387-812500；815269-815389；829265-829501；843214-843380；891883-892011；940643-941226；943518-943706；949483-949617；954955-955152；956269-956377；983374-983677；1022461-1022588；1042149-1042267；1044639-1044745；1046976-1047115；1138272-1138471；1138999-1139270；1139864-1139997；1148449-1148570；1188392-1188652；1188981-1189154；1227449-1227729；1373404-1373684；1379901-1380064；1520095-1520242；1537143-1537266；1538778-1538956；1542139-1542284；1543535-1543850；1559561-1559711；1562050-1562211；1562945-1563104；1563588-1563844；1594919-1595074；1600893-1601027；1604405-1604790；1608351-1608594；1625223-1625615；1638117-1638270；1642316-1642812；1801062-1801327；1802984-1803161；1803393-1803513；1804705-1805046；1807244-1807998；1808506-1808863；2062976-2063086；2820399-2820559；2929994-2930260；3043723-3043855；3312792-3312904；3417481-3418229；3418577-3418845；3468150-3468291；3478232-3478441；3514736-3514845；3577650-3577769；3681485-3681641；3748441-3748701；3897608-3897872；4133914-4134060；4228200-4228343；4670855-4670986；4864487-4864600；4867099-4867212；4868385-4868608；5021655-5021783；5458476-5458806；5972022-5972228；6010010-6010130；6024855-6025011；6052421-6052534；6955850-6956110；6974087-6974197；7246058-7246187；7374559-7374701；7395407-7395621；7430863-7430967；7435283-7435464；7485936-7486043；7502841-7502952；7593377-7593525；7647724-7647930；7665769-7665911；7677795-7677935；7735266-7735375；7738879-7738994；7769785-7769909；7967226-7967390；7986329-7986435；8024963-8025084；8070911-8071027；8087108-8087365；8107659-8107871；8108168-8108324；8127171-8127347；8154059-8154167；8373420-8373580；8374299-8374486；8376721-8377196；8412735-8412891；8587077-8587184；8602797-8603057；8692749-8692927；9104452-9104724；12224977-12225120；12461334-12461470；12539817-12539991；12611058-12611321；13536290-13536425；17768736-17768911；20180710-20180836；22601392-22601512；25507143-25507403；25677983-25678180；26065540-26065976；30722129-30722345；34337439-34337587；43084455-43084573；43849075-43849207；44449664-44449842；45422038-45422190；45459414-45459554；45569178-45569319；47427773-47427934；48012655-48012908；48988075-48988361；57178102-57178244；57458900-57459016；57522762-57522952；63413896-63414008；67725717-67725857；73742844-73742985；74223403-74223544；77869491-77869608；78077213-78077358；78387614-78387732；78491885-78492034；78492893-78493008；85403016-85403168；85404070-85404193；87857485-87857701；91760087-91760258；101726769-101726907；105504080-105504190；110267625-110267741；113333019-113333309；116121422-116121527；116600009-116600121；118034764-118034912；118083661-118083781；122853739-122853986；124468791-124468926；132649265-132649525；138654749-138655195；139009765-139010429；139137951-139138211；140201132-140201540；140656793-140656957；142526619-142527043；146823923-146824037；147561948-147562082；154179298-154179410；163124587-163124852；163660909-163661039；174429370-174429691；174445142-174445451；184828340-184828474；184908797-184908920；188140079-188140439；188538704-188538807；190731588-190732233
5号染色体基因组区域：
370221-370333；403141-403307；434905-435106；435326-435495；476650-476910；488444-488709；491247-491443；497290-497436；497884-498297；499493-499615；500090-500223；504490-504828；539880-539984；555877-555998；570608-570712；669672-669959；676565-676669；676942-677170；759353-759788；772459-772576；777580-777771；781019-781133；846757-846914；851149-851370；859730-859842；962016-962151；962947-963096；980707-980842；1019494-1019595；1034298-1034490；1051195-1051455；1053987-1054165；1069336-1069475；1073757-1073996；1075454-1075628；1080180-1080642；1089506-1089655；1097665-1097811；1108686-1108804；1120411-1120579；1144919-1145077；1154593-1154863；1179133-1179277；1217145-1217405；1241164-1241348；1243809-1243949；1253658-1253926；1254254-1254729；1256945-1257205；1266432-1266640；1282811-1283106；1283825-1284063；1284558-1284692；1291444-1291586；1294067-12942 78；1299499-1299649；1460382-1460534；1466228-1466358；1494976-1495154；1513626-1514220；1548911-1549171；1728964-1729226；1744869-1744986；1828054-1828171；1836672-1836932；1865373-1865566；1866120-1866464；1922890-1923026；1948908-1949168；2094934-2095046；2128984-2129119；2137148-2137300；2137569-2137674；2289160-2289288；2418606-2418866；2658898-2659158；2738445-2738712；2755905-2756178；3225839-3225962；3285954-3286311；3764218-3764375；4837640-4837769；4865961-4866225；6668876-6669007；9066642-9066792；9118929-9119249；14459296-14459397；20398668-20398841；27120803-27120919；28809975-28810091；28810272-28810498；31076623-31076758；32522822-32522953；33502556-33502889；35508308-35508554；35531828-35531966；37249949-37250092；38134910-38135176；41968320-41968580；42894344-42894448；42951511-42951795；43280263-43280428；50260006-50260205；50262673-50262796；50673089-50673308；50674046-50674175；50674745-50674885；50675061-50675270；50678374-50678509；53814624-53814941；54826510-54826825；60039895-60040165；71475162-71475304；72593842-72594009；74426799-74426971；76248710-76248824；78281663-78281964；84125258-84125409；84461326-84461456；89351908-89352029；97747125-97747229；102087695-102087875；107778016-107778142；111541377-111541637；112410762-112410874；114335370-114335475；122426703-122426804；126210914-126211206；127872000-127872262；129969721-129969860；130899817-130899926；132158772-132158986；134364413-134364714；134374532-134374648；134386361-134386497；135416286-135416613；135692492-135692752；137225296-137225468；137577519-137577796；139089776-139089946；140175775-140176602；140209246-140209798；140215809-140216109；140222067-140222587；140229326-140229782；140237294-140237554；140242111-140242471；140242621-140242939；140249614-140250359；140256898-140257234；140262975-140263499；140559126-140559422；140559441-140559818；140595239-140595499；140751473-140751609；140769104-140769223；140787768-140787883；140812344-140812479；140821469-140821682；140864470-140864835；142940925-142941044；146086086-146086288；149339730-149339831；149900917-149901065；150052308-150052409；153994606-153994714；154071013-154071134；160593123-160593263；166211722-166211934；168725398-168725501；169935196-169935315；170737929-170738059；170738221-170738463；170742243-170742346；170742603-170742734；170881742-170881877；171094976-171095266；171534695-171534880；171880461-171880593；172110491-172110905；172287915-172288022；172314099-172314250；172332444-172332739；172753613-172753803；173072299-173072442；173930868-173930977；174911383-174911503；175105255-175105356；175793369-175793484；176797875-176798065；176916394-176916502；178004056-178004247；178488076-178488213；179059433-179059542；179448715-179449072；179554390-179554594；179588619-179588897；179597289-179597486；180018437-180018714；180055772-180056044；180486604-180486734
6号染色体基因组区域：
292330-292590；564040-564158；761168-761384；1003609-1003721；1384522-1384642；1608558-1608733；1620909-1621221；2415766-2415898；2870494-2870754；3752748-3752900；4932089-4932233；5999283-5999395；6002471-6002692；6003194-6003447；7051394-7051497；7142058-7142187；7468918-7469042；7992526-7992639；9024342-9024728；10074891-10075499；10381714-10382088；10884036-10884170；12164223-12164329；12718467-12718569；14924982-14925093；16306000-16306212；17016205-17016536；19807335-19807617；19854566-19854713；21664520-21664625；25777056-25777187；26556845-26556950；26757580-26757749；27648725-27648929；27798857-27799039；28437369-28437476；28557290-28557550；28584035-28584289；28785007-28785143；29407727-29407990；29427451-29427610；29577110-29577223；29758526-29758643；29894304-29894628；30080642-30080782；30458135-30458278；30684202-30684406；30698749-30698905；30711035-30711165；31026020-31026216；31089156-31089294；31590571-31590674；31646235-31646378；31688079-31688229；31868846-31868950；32039767-32039890；32121232-32121368；32134604-32134826；32407570-32407715；32552322-32552582；32975865-32976137；33173307-33173501；33995914-33996039；34966649-34966954；35181113-35181423；35466163-35466268；37105288-37105431；37617920-37618178； 39271471-39271665；41621104-41621261；42109127-42109267；44695291-44695667；44696001-44696189；45784346-45784613；52288648-52288944；56406084-56406262；62284300-62284530；72113268-72113417；73867587-73867847；74104455-74104607；84055878-84056029；85476839-85476975；94128042-94128342；100894396-100894656；100895575-100895775；100913764-100913908；100917211-100917395；101847642-101847803；103369396-103369618；106582561-106582669；113010622-113011049；114181402-114181620；117869003-117869393；123056029-123056133；131147799-131147923；133036545-133036652；134210666-134210807；137814635-137814803；138200265-138200412；138426124-138426272；149751431-149751540；152957120-152957258；155684409-155684549；157931822-157931933；158404036-158404197；159067622-159067817；159279473-159279753；159290230-159290374；161354447-161354587；162472302-162472467；164092506-164092700；164092861-164093024；164241262-164241551；164340373-164340633；164393360-164393468；164520780-164520881；166825892-166826009；166836754-166836896；166858138-166858257；167490869-167490981；167589273-167589384；167632872-167633016；167792185-167792288；167794149-167794372；168120772-168120891；168467456-168467560；168501905-168502060；168672714-168672876；168677465-168677754；168684722-168684898；168768040-168768163；169238138-169238268；169423372-169423494；169637751-169637862；169717941-169718084；170454151-170454288；170492184-170492500
7号染色体基因组区域：
386698-386958；596151-596304；612603-612753；807815-807943；948962-949230；960114-960605；975034-975156；977023-977137；998967-999165；1039512-1040386；1052734-1052862；1054350-1054504；1108007-1108127；1216516-1216648；1254214-1254357；1303419-1303573；1325729-1325862；1363532-1363643；1370450-1370612；1428796-1428958；1443617-1443735；1560223-1560628；1687827-1687948；1715419-1715520；1746752-1746930；1778672-1778934；1865481-1865642；2040728-2040921；2057276-2057403；2185785-2185923；2293292-2293408；2349667-2349786；2473417-2473750；2499663-2499782；2561028-2561141；2681352-2681512；2700602-2700707；2770756-2771231；2800689-2800825；2801053-2801398；2801900-2802117；2802511-2802697；2854465-2854688；2855624-2855731；2859429-2859553；2859576-2859698；3849467-3849603；4030594-4030700；4049514-4049729；4050653-4050894；4118483-4118682；4184057-4184254；4187641-4187786；4839729-4839880；4850050-4850304；4855828-4855955；4856863-4857088；5340275-5340607；5389025-5389305；5400678-5401127；5458718-5458848；5521533-5521665；6188810-6188937；6194000-6194260；6438643-6438745；9661286-9661602；9765690-9765800；12610787-12610904；16890861-16890974；19184228-19184331；20823895-20824145；27153159-27153314；27183259-27183370；27185135-27185252；27203958-27204179；27206076-27206237；27309236-27309346；29186889-29187107；29229739-29229873；29233467-29233587；30635761-30635890；32802377-32802508；32802682-32802783；33080496-33080616；37531572-37531835；37888044-37888257；38278803-38278904；38468984-38469109；39393472-39393621；42267257-42267625；43484502-43485093；44153233-44153493；44801600-44801703；47576326-47576577；49815383-49815561；50518498-50518648；50633078-50633211；55134100-55134245；55146393-55146495；55812549-55812684；56949957-56950241；57715253-57715525；57929124-57929229；61821631-61821737；61822294-61822423；62574570-62574830；63020749-63021014；63642401-63642818；63643156-63643585；65509385-65509529；65969911-65970038；68000505-68000693；69923828-69923959；70252256-70252421；70502616-70502736；71202422-71202572；71407188-71407289；71876953-71877084；72813873-72814014；73157280-73157390；73245402-73245730；73819738-73819867；73843721-73843828；79368471-79368717；86974636-86974834；87105172-87105432；88119761-88119870；90894982-90895101；92238086-92238355；96626903-96627103；96650566-96650732；97912540-97912641；97978607-97978735；98424288-98424446；99017370-99017475；99517460-99517584；99723100-99723311；100203033-100203651；100304644-100304922；100638850-100639004；100643545-100643783；100769873-100769978；100845605-100845856；100882954-100883078；100921843-100921974；101839163-101839411；101847656-101847785；101848202-101848356；102098181-102098322；104445585-104445689；107592541-107592723；108108891-108109021；111381499-111381600；113717 270-113717419；114561714-114562037；120967900-120968043；127991181-127991655；128494130-128494305；128797855-128797964；130127586-130127846；130130122-130130384；131477409-131477517；131831439-131831581；134233790-134233937；134918382-134918642；134931024-134931636；134932449-134932808；138440463-138440653；139167426-139167533；139256205-139256604；145057765-145057874；146731529-146731630；148768321-148768581；149318385-149318558；149410858-149410996；149411103-149411395；149416720-149416880；150021487-150021599；150074779-150074900；150711033-150711221；150715107-150715221；150778880-150779089；150811280-150811444；150820337-150820597；150871185-150871461；150871479-150872017；151077325-151077428；151145267-151145596；151300423-151300705；151393406-151393666；151433279-151433395；151546368-151546484；151553416-151553551；153108577-153108678；153446852-153446954；154667753-154667907；155006030-155006290；155010884-155011005；155191150-155191256；155325649-155326005；155674685-155675066；155996484-155996598；156733725-156734217；156734411-156734671；156796837-156797243；156798175-156798342；156804311-156804448；156810954-156811214；157095169-157095284；157195506-157195725；157207071-157207348；157219644-157219796；157233133-157233247；157333722-157333853；157335384-157335700；157347662-157347854；157355247-157355522；157408049-157408189；157413991-157414251；157441452-157441590；157448993-157449114；157474919-157475065；157483305-157483509；157543646-157543806；157601991-157602288；157678926-157679078；157694287-157694547；157849665-157849811；157860362-157860560；158107343-158107491；158107804-158108211；158109335-158109483；158133812-158133997；158277603-158277817；158349791-158349971
8号染色体基因组区域：
325033-325176；655191-655314；668025-668285；748280-748404；865864-865972；914830-914945；982153-982280；1112787-1112903；1114396-1114539；1132826-1133109；1134434-1134574；1140668-1140936；1200860-1201016；1263493-1263704；1276872-1277004；1385178-1385371；1472152-1472285；1553170-1553377；1553585-1553699；1639908-1640017；1779776-1779917；1821298-1821488；1829774-1830034；1911528-1911635；1960222-1960370；2003769-2003919；2014314-2014500；2041819-2041946；2143616-2143777；2196631-2196817；3316846-3316986；3855558-3855687；6420228-6420539；6637732-6637976；6930518-6930648；8639032-8639184；8639785-8640185；10192033-10192615；10339377-10339517；10459836-10459961；10589192-10589310；10755723-10755878；11302092-11302294；16459664-16460187；17767417-17767677；21970244-21970385；22034616-22034894；22844224-22844507；22928561-22928662；23423848-23424025；23559923-23560087；23564119-23564592；24918754-24918890；27327223-27327527；33423227-33423336；35979074-35979247；37655351-37655503；37688979-37689092；37699311-37699479；37743949-37744084；38411582-38411683；52183857-52184049；53852210-53852328；55533664-55533924；56363638-56363809；57350958-57351068；57361036-57361318；58114805-58114915；58120340-58120489；58122875-58123032；58123194-58123375；58124249-58124374；58125408-58125615；58129539-58129651；61326287-61326429；62823811-62823937；65488536-65488647；72917233-72917352；73848457-73848579；74197864-74198042；76576769-76576918；76951131-76951259；76971864-76971967；77108661-77108787；77359751-77359908；81523895-81524062；89340637-89340788；89688296-89688432；90624033-90624163；95962213-95962411；96085465-96085584；97340180-97340319；99961365-99961623；102202802-102203072；115323639-115323782；116048978-116049122；116170821-116170926；117962271-117962510；119964253-119964486；124730010-124730213；126709638-126709751；128930104-128930240；128950431-128950534；130641180-130641350；132752295-132752443；133141655-133141779；135490693-135490815；138135392-138135499；139096462-139096585；142185359-142185494；142233347-142233574；142238956-142239142；142302106-142302250；142310897-142311126；142350518-142350647；142437818-142437936；142981762-142981869；143129202-143129350；143150270-143150420；143182559-143182689；143203588-143203866；143207541-143207779；143214216-143214507；143237807-143237910；143254264-143254468；143356856-143357002；143378932-143379065；143405 741-143405872；143407806-143408066；143472969-143473155；143546760-143546877；143560731-143561040；143582378-143582484；143609379-143609638；143614730-143614910；143620631-143620753；143623289-143623631；143625581-143626108；143645492-143645722；143781627-143781955；143781960-143782226；143879967-143880112；144140196-144140301；144163515-144163721；144238672-144238779；144262756-144263033；144296873-144297020；144343806-144343929；144358407-144358582；144361213-144361341；144680983-144681132；144801025-144801264；144815220-144815359；144820742-144820893；144965127-144965255；144990294-144990407；145002793-145002935；145011866-145012151；145045850-145045956；145048324-145048432；145051528-145051937；145107745-145107846；145111331-145111500；145164790-145164936；145551082-145551217；145747723-145747832；145749775-145750137；146232962-146233084
9号染色体基因组区域：
4297532-4297663；4298373-4298504；4662863-4662987；6328354-6328646；8835157-8835270；11197185-11197346；19102622-19102747；22433833-22434003；33386385-33386526；34457129-34457379；34623938-34624080；43134591-43134844；66837800-66837982；67340024-67340186；72046230-72046367；72131425-72131587；78977770-78977894；84228185-84228387；87903840-87904107；94860379-94860687；97349536-97349722；97807640-97807900；97865073-97865232；98477893-98478005；99463426-99463572；100614849-100614956；106761967-106762082；106964064-106964360；107091714-107091823；107125513-107125642；109263886-109264032；111617732-111617992；112296520-112296746；114090182-114090286；116663391-116663558；116930110-116930382；122631840-122631944；124499750-124499937；126154320-126154556；126776221-126776323；127071526-127071681；127600928-127601523；127623377-127623523；129100371-129100602；130700840-130701068；131156805-131156923；132016264-132016381；132253313-132253506；132481611-132481764；133767796-133767925；133813518-133813707；135374804-135374905；136501744-136501893；137111294-137111435；137620170-137620706；137677929-137678047；137996213-137996370；138135144-138135249；138156801-138156937；138193645-138193784；138330182-138330329；138393583-138393685；138408073-138408187；138441301-138441433；138587706-138587829；138591456-138591568；138660867-138661107；138666312-138666525；138670628-138670837；138672837-138673181；138674560-138674856；138680030-138680285；138836905-138837056；138838026-138838170；138839522-138840012；139048246-139048499；139143521-139143695；139146527-139146662；139250789-139250973；139515702-139515832；139565084-139565239；139734956-139735106；139840431-139840679；139907612-139907775；140126911-140127171；140136997-140137136；140174189-140174312；140217992-140218215；140329560-140329670；140419847-140419983；140501304-140501413；140777297-140777579；140795348-140795503；140944063-140944202；141007494-141007680；141042575-141042717
X染色体基因组区域：
7896030-7896163；22049899-22050181；22290873-22290977；27999541-27999771；30233607-30233727；31089585-31089704；37351147-37351407；39951483-39951665；40439815-40440129；41334218-41334386；48367092-48367580；55115535-55115681；56057985-56058125；63264016-63264129；64627133-64627568；66766042-66766302；70128510-70128671；73619264-73619532；79082990-79083123；84634307-84634451；99663831-99664091；101397267-101397579；114425893-114426012；124824739-124824842；129039937-129040067；131543695-131543825；133306625-133306958；135229594-135230119；135962416-135962568；141291476-141291746；142840351-142840728；147582015-147582583；150151693-150152077；152907700-152908275；152909455-152909751；153033217-153033347；153695647-153695785；154522076-154522200；154624596-154624745

Claims (51)

  1. 一种检测待测样品中的至少一个特定人类基因组区域上的、携带特定的DNA甲基化修饰或特定的DNA甲基化单倍型的试剂在制备用于检测、监测或预测受试者是否患有胎盘源性妊娠期病症的试剂或试剂盒中的用途；其中，所述特定的DNA甲基化修饰或所述特定的DNA甲基化单倍型来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域。
  2. 根据权利要求1所述的用途，其中，所述待测样品来源于所述受试者；
    所述甲基化差异区域为如附表1所示的基因组区域；
    所述特定的DNA甲基化修饰为如附表1所示的基因组区域内特定CpG位点的甲基化修饰；
    所述特定的甲基化单倍型为如附表1所示的基因组区域所覆盖的DNA甲基化单倍型；
    所述特定的DNA甲基化修饰或特定的甲基化单倍型能区分正常妊娠孕妇胎盘和患有胎盘源性妊娠期病症的孕妇胎盘。
  3. 根据权利要求1-2任一项所述的用途，其中，所述待测样品来源于所述受试者；
    所检测的所述特定的DNA甲基化修饰或特定的DNA甲基化单倍型位于选自如下(i)-(iii)所示的区域中的一个或多个区域：
    (i)位于LTR12转座子家族区域；优选的，所述LTR12转座子为LTR12C转座子或LTR12E转座子；
    (ii)位于FLT1基因及其调控区域；优选的，所述FLT1基因及其调控区域为人类染色体chr13:28800000bp-302000000bp之间的区域；
    (iii)位于LIFR基因及其调控区域；优选的，所述LIFR基因及其调控区域为人类染色体chr5:38370000bp–38840000bp之间的区域。
  4. 根据权利要求1-3任一项所述的用途，其中，所述待测样品选自来源于血液、尿液、粪便、唾液、口腔拭子、宫颈分泌物、宫颈涂片、羊水穿刺、胎儿绒毛或胎儿循环细胞的样品；优选的，所述血液为外周血，更优选的，所述外周血为血浆。
  5. 根据权利要求1-4任一项所述的用途，其中，胎盘源性妊娠期病症选自妊娠期糖尿病、双胎输血综合征、胎儿生长受限、妊娠期高血压、子痫前期、重度子痫前期、继发型子痫前期、非典型子痫前期、HELLP综合症中的一种或多种；优选的，所述妊娠期病症选自子痫前期、重度子痫前期、继发型子痫前期、非典型子痫前期中的一种或多种。
  6. 根据权利要求1-5任一项所述的用途，其中，检测所述来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域的试剂选自DNA甲基化状态显示剂；或
    检测来自受试者样品中DNA甲基化修饰的存在的试剂；
    优选的，所述检测来自受试者样品中DNA甲基化修饰的存在的试剂为富集特征DNA所需的试剂。
  7. 根据权利要求6所述的用途，其中，所述DNA甲基化状态显示剂选自识别甲基化DNA的抗体或结合蛋白、亚硫酸氢盐、具有DNA催化氧化作用的酶类、具有DNA脱氨基作用的酶类或甲基化敏感酶，或其组合；优选的，所述甲基化敏感酶选自甲基化敏感的限制性内切酶，更优选的，所述甲基化敏感的限制性内切酶选自HpaII或BstUI，或其组合。
  8. 根据权利要求6所述的用途，其中，所述富集特征DNA所需的试剂选自杂交探针捕获法所需的试剂、聚合酶链式反应扩增法所需的试剂、锚定核酸扩增法所需的试剂、用于边合成边测序检测的试剂，或用于单分子测序的试剂，或其组合。
  9. 根据权利要求8所述的用途，其中，所述富集特征DNA所需的试剂为探针或探针组；优选的，所述探针或探针组为EP-007探针或探针组。
  10. 根据权利要求1-9任一项所述的用途，其中，所述检测为产前检测；优选的，所述产前检测为无创产前检测；更优选的，所述产前检测为孕早期无创产前检测。
  11. 根据权利要求1-10任一项所述的用途，其中，所述DNA为gDNA或cfDNA。
  12. 一种试剂盒，其用于检测待测样品中至少一个特定的DNA甲基化修饰或特定的DNA甲基化单倍型，以检测、监测或预测受试者是否患有胎盘源性妊娠期病症，所述试剂盒包含：
    (a)检测DNA甲基化修饰的存在的试剂；
    可选的，所述试剂盒还可以包含：
    (b)检测DNA甲基化状态的显示剂；
    其中，所述特定的DNA甲基化修饰或特定的DNA甲基化单倍型来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域。
  13. 根据权利要求12所述的试剂盒，其中，所述甲基化差异区域为如附表1所示的基因组区域；
    所述特定的DNA甲基化修饰为如附表1所示的基因组区域内特定CpG位点的甲基化修饰；
    所述特定的甲基化单倍型为如附表1所示的基因组区域所覆盖的DNA甲基化单倍型；
    所述特定的DNA甲基化修饰或特定的甲基化单倍型能区分正常妊娠孕妇胎盘和患有胎盘源性妊娠期病症的孕妇胎盘。
  14. 根据权利要求12-13任一项所述的试剂盒，其中，所述检测DNA甲基化修饰的存在的试剂为富集特征DNA所需的试剂；优选的，所述富集特征DNA所需的试剂为探针或探针组；更优选的，所述探针或探针组为EP-007探针或探针组。
  15. 根据权利要求12-14任一项所述的试剂盒，其中，所述DNA甲基化状态显示剂选自识别甲基化DNA的抗体或结合蛋白、亚硫酸氢盐、具有DNA催化氧化作用的酶类、具有DNA脱氨基作用的酶类或甲基化敏感酶，或其组合；优选的，所述甲基化敏感酶选自甲基化敏感的限制性内切酶，更优选的，所述甲基化敏感的限制性内切酶选自HpaII或BstUI，或其组合。
  16. 根据权利要求12-15任一项所述的试剂盒，其中，所述待测样品选自来源于血液、尿液、粪便、唾液、口腔拭子、宫颈分泌物、宫颈涂片、羊水穿刺、胎儿绒毛或胎儿循环细胞的样品；优选的，所述血液为外周血，更优选的，所述外周血为血浆。
  17. 根据权利要求12-16任一项所述的试剂盒，其中，所述检测为产前检测；优选的，所述产前检测为无创产前检测；更优选的，所述产前检测为孕早期无创产前检测。
  18. 根据权利要求12-17任一项所述的试剂盒，其中，所述DNA为gDNA或cfDNA。
  19. 一种多核苷酸，所述多核苷酸可以：
    (i)在高严格性杂交条件或非常高严格性杂交条件下，与EP-007探针或探针组所示的多核苷酸杂交；或
    (ii)具有与(i)所示的多核苷酸的序列的反向互补序列；
    其中，所述多核苷酸用于检测样品中至少一个特定的DNA甲基化修饰或特定的DNA甲基化单倍型。
  20. 如权利要求19所述的多核苷酸，其与EP-007探针或探针组所示的核苷酸序列或其反向互补序列具有至少90％，可选至少95％，优选至少97％，更优选至少98％，最优选至少99％的序列同一性的序列。
  21. 如权利要求19或20所述的多核苷酸，其中，所述多核苷酸的序列包含如EP-007探针或探针组所示的核苷酸序列；优选的，所述多核苷酸的序列为如EP-007探针或探针组所示的核苷酸序列。
  22. 如权利要求19或20所述的多核苷酸，其中，所述多核苷酸为DNA探针。
  23. 一种检测待测样品中是否存在至少一个特定人类基因组区域上的，具有胎盘源性妊娠期病症特征的DNA片段的方法，所述方法包括检测步骤，所述检测步骤用于检测：
    (a)待测样品中是否存在至少一种胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰或其修饰丰度；或
    (b)待测样品中是否存在至少一种胎盘源性妊娠期病症特征的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度；
    其中，所述胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰或所述DNA甲基化单倍型来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域。
  24. 根据权利要求23所述的方法，其中，所述胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰发生在如附表1所示的基因组区域所覆盖的位点上；
    所述DNA甲基化单倍型为如附表1所示的基因组区域所覆盖的DNA甲基化单倍型。
  25. 根据权利要求23-24任一项所述的方法，其中，所检测的所述特定的DNA甲基化修饰或特定的DNA甲基化单倍型位于选自如下(i)-(iii)所示的区域中的一个或多个区域：
    (i)位于LTR12转座子家族区域；优选的，所述LTR12转座子为LTR12C转座子或LTR12E转座子；
    (ii)位于FLT1基因及其调控区域；优选的，所述FLT1基因及其调控区域为人类染色体chr13:28800000bp-302000000bp之间的区域；
    (iii)位于LIFR基因及其调控区域；优选的，所述LIFR基因及其调控区域为人类染色体chr5:38370000bp–38840000bp之间的区域。
  26. 根据权利要求23-25任一项所述的方法，其通过检测DNA甲基化修饰的存在的试剂对所述(a)或(b)进行检测，其中，所述检测DNA甲基化修饰的存在的试剂为富集特征DNA所需的试剂；优选的，所述富集特征DNA所需的试剂为探针或探针组；更优选的，所述探针或探针组为EP-007探针或探针组。
  27. 根据权利要求23-26任一项所述的方法，其中，所述方法还包括从待测样品中提取DNA的步骤，所述待测样品为外周血样品；优选的，所述外周血样品为血浆样品。
  28. 根据权利要求23-27任一项所述的方法，其中，所述DNA为gDNA或cfDNA。
  29. 一种检测、监测或预测受试者是否患有胎盘源性妊娠期病症的方法，所述方法包括下列步骤：
    (1)检测步骤：
    (a1)检测待测样品中是否存在至少一种胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰或其修饰丰度，所述胎盘源性妊娠期病症特征的DNA甲基化修饰来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域；或
    (b1)检测待测样品中是否存在胎盘源性妊娠期病症特征的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度，所述DNA甲基化单倍型来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域；
    (2)比较步骤：
    (a2)比较所述待测样品的特定CpG位点的甲基化修饰或其修饰丰度的检测结果和具有或不具有胎盘源性妊娠期病症的胎盘样本的特定CpG位点的甲基化修饰或其修饰丰度的检测结果；或
    (b2)比较所述待测样品的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度的检测结果和具有或不具有胎盘源性妊娠期病症的胎盘样本的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度的检测结果；
    (3)判断步骤：
    (i)如果(a2)或(b2)中，所述待测样品和不具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较低或相似度统计量值显著低于预期，或者所述待测样品和具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较高或相似度统计量值显著高于预期，那么所述受试者患有胎盘源性妊娠期病症；
    (ii)如果(a2)或(b2)中，所述待测样品和不具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较高或相似度统计量值显著高于预期，或者所述待测样品和具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较低或相似度统计量值显著低于预期，那么所述受试者未患有胎盘源性妊娠期病症；
    其中，所述待测样品来源于所述受试者。
  30. 根据权利要求29所述的方法，其中，所述胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰发生在如附表1所示的基因组区域所覆盖的位点上；所述DNA甲基化单倍型为如附表1所示的基因组区域所覆盖的DNA甲基化单倍型。
  31. 根据权利要求29-30任一项所述的方法，其中，所检测的所述特定CpG位点的甲基化修饰或特定的DNA甲基化单倍型位于选自如下(i)-(iii)所示的区域中的一个或多个区域：
    (i)位于LTR12转座子家族区域；优选的，所述LTR12转座子为LTR12C转座子或LTR12E转座子；
    (ii)位于FLT1基因及其调控区域；优选的，所述FLT1基因及其调控区域为人类染色体 chr13:28800000bp-302000000bp之间的区域；
    (iii)位于LIFR基因及其调控区域；优选的，所述LIFR基因及其调控区域为人类染色体chr5:38370000bp–38840000bp之间的区域。
  32. 根据权利要求29-31任一项所述的方法，其中，所述胎盘源性妊娠期病症选自妊娠期糖尿病、双胎输血综合征、胎儿生长受限、妊娠期高血压、子痫前期、重度子痫前期、继发型子痫前期、非典型子痫前期、HELLP综合症中的一种或多种；优选的，所述妊娠期病症选自子痫前期、重度子痫前期、继发型子痫前期、非典型子痫前期中的一种或多种。
  33. 根据权利要求29-32任一项所述的方法，其中，所述胎盘源性妊娠期病症的特异甲基化区域包括超甲基化区域和低甲基化区域。
  34. 根据权利要求29-33任一项所述的方法，其中，步骤(3)中所采用的相似度统计量判断方法选自：相关分析(correlation)、t检验、Z检验、超几何检验、傅里叶分析(Fourier analysis)、小波分析(Wavelet analysis)、主成分分析(PCA)、流形嵌入(tSNE)、正交矩阵分解(NMF)、支持向量机(SVM)、K-最近邻(KNN)、K-均值(k-means)、线性回归(LM)、广义线性回归(GLM)、混合高斯模型(GMM)、神经网络(NN)、随机森林(RF)、自编码网络(Autoencoder)、深度神经网络(DNN)及其衍生变体。
  35. 根据权利要求29-34任一项所述的方法，其中，通过检测DNA甲基化修饰的存在的试剂，检测特定CpG位点的甲基化修饰或其修饰丰度；或检测特定的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度。
  36. 根据权利要求35所述的方法，其通过检测DNA甲基化修饰的存在的试剂进行检测，其中，所述检测DNA甲基化修饰的存在的试剂为富集特征DNA所需的试剂；优选的，所述富集特征DNA所需的试剂为探针或探针组；更优选的，所述探针或探针组为EP-007探针或探针组。
  37. 根据权利要求29-36任一项所述的方法，其中，所述方法还包括从待测样品中提取DNA的步骤，所述待测样品为外周血样品；优选的，所述外周血样品为血浆样品。
  38. 根据权利要求37所述的方法，其中，所述DNA为gDNA或cfDNA。
  39. 根据权利要求29-38任一项所述的方法，其中，所述方法进一步包括富集所述DNA或对所述DNA进行测序的步骤。
  40. 一种用于检测、监测或预测受试者是否患有胎盘源性妊娠期病症的设备，包括：
    处理器；
    用于存储处理器和执行指令的存储器；
    其中，所述处理器被配制为在执行所述处理器可执行指令时，实现权利要求23-39任一项所述的方法。
  41. 一种非易失性计算机可读存储介质，其上存储有计算机程序指令，所述计算机程序指令被处理器执行时，实现权利要求23-39任一项所述的方法。
  42. 一种检测待测样品中至少一个特定CpG位点的特定甲基化修饰或至少一种DNA甲基化单倍型的系统，所述系统包括检测模块，所述检测模块用于检测：
    (a)待测样品中是否存在至少一种胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰或其修饰丰度；或
    (b)待测样品中是否存在至少一种胎盘源性妊娠期病症特征的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度；
    其中，所述胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰或所述DNA甲基化单倍型来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域。
  43. 根据权利要求42所述的方法，其中，所述胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰发生在如附表1所示的基因组区域所覆盖的位点上；所述DNA甲基化单倍型为如附表1所示的基因组区域所覆盖的DNA甲基化单倍型。
  44. 根据权利要求42-43任一项所述的系统，其中，所检测的所述特定的DNA甲基化修饰或特定 的DNA甲基化单倍型位于选自如下(i)-(iii)所示的区域中的一个或多个区域：
    (i)位于LTR12转座子家族区域；优选的，所述LTR12转座子为LTR12C转座子或LTR12E转座子；
    (ii)位于FLT1基因及其调控区域；优选的，所述FLT1基因及其调控区域为人类染色体chr13:28800000bp-302000000bp之间的区域；
    (iii)位于LIFR基因及其调控区域；优选的，所述LIFR基因及其调控区域为人类染色体chr5:38370000bp–38840000bp之间的区域。
  45. 一种检测、监测或预测受试者是否患有胎盘源性妊娠期病症的系统，所述系统包括下列模块：
    (1)检测模块，所述检测模块检测：
    (a1)待测样品中是否存在至少一种胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰或其修饰丰度，所述胎盘源性妊娠期病症特征的DNA甲基化修饰来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域；或
    (b1)待测样品中是否存在胎盘源性妊娠期病症特征的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度，所述DNA甲基化单倍型来源于正常妊娠孕妇胎盘和患有胎盘源性妊娠期疾病孕妇胎盘的甲基化差异区域；
    (2)比较模块，所述比较模块比较：
    (a2)比较所述待测样品的特定CpG位点的甲基化修饰或其修饰丰度的检测结果和具有或不具有胎盘源性妊娠期病症的胎盘样本的特定CpG位点的甲基化修饰或其修饰丰度的检测结果；或
    (b2)比较所述待测样品的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度的检测结果和具有或不具有胎盘源性妊娠期病症的胎盘样本的DNA甲基化单倍型或所述DNA甲基化单倍型的丰度的检测结果；
    (3)判断模块，所述判断模块判断受试者是否患有胎盘源性妊娠期病症：
    (i)如果(a2)或(b2)中，所述待测样品和不具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较低或相似度统计量值显著低于预期，或者所述待测样品和具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较高或相似度统计量值显著高于预期，那么所述受试者患有胎盘源性妊娠期病症；
    (ii)如果(a2)或(b2)中，所述待测样品和不具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较高或相似度统计量值显著高于预期，或者所述待测样品和具有所述胎盘源性妊娠期病症的胎盘样本的检测结果相似度较低或相似度统计量值显著低于预期，那么所述受试者未患有胎盘源性妊娠期病症。
  46. 根据权利要求45所述的系统，其中，所述胎盘源性妊娠期病症特征的特定CpG位点的甲基化修饰发生在如附表1所示的基因组区域所覆盖的位点上；所述DNA甲基化单倍型为如附表1所示的基因组区域所覆盖的DNA甲基化单倍型。
  47. 根据权利要求45-46任一项所述的系统，其中，所检测的所述特定CpG位点的甲基化修饰或特定的DNA甲基化单倍型位于选自如下(i)-(iii)所示的区域中的一个或多个区域：
    (i)位于LTR12转座子家族区域；优选的，所述LTR12转座子为LTR12C转座子或LTR12E转座子；
    (ii)位于FLT1基因及其调控区域；优选的，所述FLT1基因及其调控区域为人类染色体chr13:28800000bp-302000000bp之间的区域；
    (iii)位于LIFR基因及其调控区域；优选的，所述LIFR基因及其调控区域为人类染色体chr5:38370000bp–38840000bp之间的区域。
  48. 根据权利要求23-39任一项所述的方法，其中，所述方法包含高通量测序。
  49. 根据权利要求23-39任一项所述的方法，其中，所述方法包含定量PCR(qPCR)或数字PCR(digital PCR)。
  50. 根据权利要求23-39任一项所述的方法，其中，所述方法包含甲基化特异PCR。
  51. 根据权利要求23-39任一项所述的方法，其中，所述方法包含甲基化敏感酶切。
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