WO2020119626A1 - Method for non-invasive prenatal testing of fetus for genetic disease - Google Patents

Method for non-invasive prenatal testing of fetus for genetic disease Download PDF

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WO2020119626A1
WO2020119626A1 PCT/CN2019/123983 CN2019123983W WO2020119626A1 WO 2020119626 A1 WO2020119626 A1 WO 2020119626A1 CN 2019123983 W CN2019123983 W CN 2019123983W WO 2020119626 A1 WO2020119626 A1 WO 2020119626A1
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dna
methylation
fetal
region
genetic disease
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张翼
刘九思
常璐媛
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贵阳优乐复生医学检验所有限公司
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    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
    • C12Q1/6869—Methods for sequencing
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present disclosure relates to a series of methylation modifications and combinations of human genomic loci that can be used for non-invasive prenatal diagnosis and screening. More specifically, the present disclosure relates to a series of combinations of methylation modification of human genomic loci that are specific to placental tissues, and can be used for non-invasive prenatal diagnosis and screening of whether or not the fetus has genetic disease or the risk of genetic disease For example, it can be used for non-invasive diagnosis and screening for alpha thalassemia. The present disclosure also relates to an experimental method for non-invasive prenatal diagnosis and screening of alpha thalassemia based on this series of genomic site methylation modifications, and related high-throughput sequencing kits.
  • Alpha-thalassaemia ( ⁇ -Thalassaemia, referred to as ⁇ -thalassaemia) is an inherited anemia caused by decreased or suppressed synthesis of hemoglobin ⁇ peptide chain due to mutation or deletion of ⁇ -globin gene.
  • Alpha-thalassaemia occurs mostly in tropical and subtropical regions, such as Southeast Asia, the Indian subcontinent, and southern China.
  • ⁇ -thalassaemia is one of the most common single-gene genetic diseases, and the carrying rate of ⁇ -thalassaemia genes is 5%. In mainland China, the carrying rate of ⁇ -thalassaemia gene is 7.88%, and it is as high as 14.13% in Guangxi province.
  • ⁇ -thalassemia The clinical manifestations of ⁇ -thalassemia are directly related to the number of ⁇ -globin genes.
  • ⁇ -globin genes When all ⁇ -globin genes are deleted (--/--, severe ⁇ -thalassemia), the patient develops fetus during the fetal period, which is manifested as severe anemia Accompanied by systemic edema and other symptoms (HbBarts'hydropsfetalis, Bartholin's fetal edema syndrome).
  • the disease is fatal to the fetus, and may cause serious or even life-threatening complications to the mother. Without adequate medical care, the maternal mortality rate may be close to 50%.
  • the genetic disease particularly places a heavy public health burden on less developed countries and regions.
  • the standard prenatal diagnosis method for ⁇ -thalassaemia is to perform genotyping screening for high-risk couples, and then to use prenatal diagnosis of placenta or fetal cells using villous biopsy, amniocentesis, etc.
  • these conventional methods are invasive, may cause damage to the fetus and pregnant women, and even have the risk of fetal abortion, while also increasing the psychological burden of pregnant women.
  • the risk of surgical abortion in villous biopsy and amniocentesis is 0.22% and 0.11%, respectively (Akolekar et al., Ultrasound Obstet Gynecol, 2015, 45(1): 16-26).
  • NIPT non-invasive prenatal testing
  • cffDNA for noninvasive prenatal diagnosis of single-gene genetic diseases such as ⁇ -thalassemia and Wilson disease also has some scientific research results (Chiu et al., Lancet 360:998-1000, 2002; Lv W et al., ClinChem 61:172–181).
  • Severe ⁇ -thalassaemia (--/--) is caused by the deletion of a long fragment of the ⁇ -globin gene (for example, the SEA subtype), and cell-free DNA (cfDNA, also known as free DNA) is highly fragmented (Lo, YMD et al. (2010) Sci. Transl. Med. 2, 61ra91). Therefore, traditional methods for detecting ⁇ -thalassaemia genes, such as gap polymerase chain reaction (Gap-PCR ), Multiple Ligation Probe Amplification Technology (MLPA), etc., are not suitable for non-invasive detection of ⁇ -thalassaemia genes.
  • Gap-PCR gap polymerase chain reaction
  • MLPA Multiple Ligation Probe Amplification Technology
  • the fetal free DNA concentration in the sample is high, with a median value of 23.41%, far exceeding the average level (Ge et al., PLoS One. 2013 ; 8(6):e67464). Therefore, in order to avoid maternal DNA interference and to more efficiently and accurately assess the genotype of the fetal ⁇ -globin gene, it is necessary to identify specific, independent of the limiting factors such as polymorphic sites of the fetal genome or fetal gender, independent Fetal DNA markers.
  • the present disclosure provides fetal DNA markers, detection methods, and detection kits that can be used for non-invasive prenatal genetic diagnosis of fetal alpha-thalassemia. Compared with the prior art, the technical solution related to the present disclosure can carry out non-invasive prenatal genetic diagnosis of fetal ⁇ -thalassemia more safely, efficiently, accurately and economically.
  • the present disclosure includes but is not limited to the following technical solutions involved.
  • the methylation mode is a single-molecule DNA methylation state; wherein, the methylation site of the single-molecule DNA methylation state is selected from the region encoding the ⁇ -globin gene; preferably, the fetal genetic disease is ⁇ -Thalassemia.
  • the region encoding the ⁇ -globin gene is selected from the region between 190000-250000 base pairs of human chromosome 16; preferably, the encoding ⁇ -globin gene Is selected from any one of the base pairs of human chromosome 16 at the following positions: 202146, 202148, 202161, 202170, 202178, 202310, 202425, 205180, 205229, 205234, 205245, 209848, 209885, 209922, 209959, 209990 ,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763 ,210800,210821,210832,210834,210837,210868,210873,210964,210974,
  • the region encoding the ⁇ -globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484, 229499, 229527 , 229535 or any combination of two or more of the above base pairs; preferably, the region encoding the ⁇ -globin gene contains at least the following positions selected from human chromosome 16 Any base pair: 229484, 229499 or a combination of the above base pairs.
  • the reagent for detecting fetal-specific DNA methylation patterns is selected from DNA methylation status indicators and/or for detecting fetal specificity in biological samples from pregnant women
  • a reagent for the presence of sexual methylation pattern DNA; preferably, the reagent for detecting the presence of fetal-specific methylation pattern DNA in the biological sample from a pregnant woman is selected from reagents required for enriching characteristic free DNA.
  • the DNA methylation status display agent is selected from an antibody or a binding protein that recognizes methylated DNA, bisulfite, and an enzyme that has catalytic oxidation of DNA , Enzymes with DNA deamination or methylation-sensitive enzymes, or a combination thereof; preferably, the methylation-sensitive enzyme is selected from methylation-sensitive restriction enzymes, more preferably, the The methylation-sensitive restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
  • reagent required for enriching characteristic free DNA is selected from reagents required for the liquid-phase hybridization probe capture method and the polymerase chain reaction amplification method.
  • sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid or cord blood, or combination.
  • test is a prenatal test; preferably, the prenatal test is a non-invasive prenatal test.
  • kits for detecting the presence or absence of fetal-specific DNA methylation patterns in biological samples from pregnant women comprising:
  • the kit further includes:
  • the fetal-specific DNA methylation mode is a single-molecule methylation state of fetal-specific DNA.
  • kits according to 9 which is used to detect whether a fetus has a genetic disease or a risk of suffering from a genetic disease; preferably, the genetic disease is ⁇ -thalassemia.
  • the methylation site of the methylation state of the single molecule of fetal-specific DNA is selected from the region encoding the ⁇ -globin gene; preferably, The region encoding the ⁇ -globin gene is selected from the region between 190000-250000 base pairs of human chromosome 16; more preferably, the region encoding the ⁇ -globin gene is selected from the following positions on human chromosome 16 Any one of the base pairs: 202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068, 210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,2106
  • the region encoding the ⁇ -globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484,229499, 229527,229535 or any combination of two or more of the above base pairs; preferably, the region encoding the ⁇ -globin gene contains at least the following positions selected from human chromosome 16 Any one of the base pairs: 229484, 229499 or a combination of the above base pairs.
  • the DNA methylation status indicator is selected from an antibody or a binding protein that recognizes methylated DNA, bisulfite, and has a catalytic oxidation of DNA Enzymes, enzymes with DNA deamination or methylation sensitive enzymes, or a combination thereof; preferably, the methylation sensitive enzyme is selected from methylation sensitive restriction enzymes, more preferably, The methylation-sensitive restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
  • reagent required for enriching characteristic free DNA is selected from reagents required for the liquid phase hybridization probe capture method, polymerase chain reaction amplification method
  • kits for detecting whether a fetus has a genetic disease or a risk of suffering from a genetic disease comprising:
  • the kit further includes:
  • the fetal-specific DNA methylation mode is a single-molecule methylation state of fetal-specific DNA.
  • the kit according to 16 which detects fetal-specific DNA methylation patterns in biological samples from pregnant women, and thereby detects whether the fetus has a genetic disease or a risk of suffering from a genetic disease; preferably, the The genetic disease is ⁇ -thalassemia.
  • the methylation site of the single-molecule methylation state is selected from a region encoding an ⁇ -globin gene; preferably, the encoding ⁇ -globin The region of the globin gene is selected from the region between 190000-250000 base pairs of human chromosome 16; more preferably, the region encoding the ⁇ -globin gene is selected from any one of the following positions on human chromosome 16 Base pair: 202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215, 210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,
  • the region encoding the ⁇ -globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484,229499, 229527,229535 or any combination of two or more of the above base pairs; preferably, the region encoding the ⁇ -globin gene contains at least the following positions selected from human chromosome 16 Any one of the base pairs: 229484, 229499 or a combination of the above base pairs.
  • sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid or cord blood, or Its combination.
  • the DNA methylation status display agent is selected from an antibody or a binding protein that recognizes methylated DNA, bisulfite, and has a catalytic oxidation of DNA Enzymes, enzymes with DNA deamination or methylation sensitive enzymes, or a combination thereof; preferably, the methylation sensitive enzyme is selected from methylation sensitive restriction enzymes, more preferably, The methylation-sensitive restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
  • kits according to any one of 16-21 wherein the reagent required for enriching characteristic free DNA is selected from reagents required for the liquid phase hybridization probe capture method, polymerase chain reaction amplification method The reagents required, the reagents required for the anchored nucleic acid amplification method, the reagents for sequencing-by-synthesis detection, or the reagents for single molecule sequencing, or a combination thereof.
  • a method for detecting whether a fetus has a genetic disease or a risk of having a genetic disease comprising the following steps:
  • Detection step detecting the DNA methylation pattern of the total DNA fragments in the treated biological sample; wherein, the biological sample is a plasma sample of a pregnant female;
  • Distinguishing step distinguish different patterns of DNA methylation of the total DNA fragments in the biological sample obtained in the detection step;
  • Calculation step Calculate the proportion of DNA fragments carrying a specific DNA methylation pattern in the biological sample to the total DNA fragments in the area, and find the DNA methylation pattern that exists only in placental tissue but not in the plasma of infertile women , That is, fetal-specific DNA methylation pattern;
  • Comparison step comparing the ratio of the DNA fragments of the pregnant women to the DNA fragments of the fetus in the gene region corresponding to the genetic disease to the total number of DNA fragments in the region and the DNA fragments of the fetus from the control gene region to the region The ratio of the total number of DNA fragments;
  • the method further includes a judgment step:
  • the fetus has a genetic disease or Have a high risk of suffering from genetic diseases;
  • the fetus does not have genetic
  • the disease may or may not have a risk of having a genetic disease or have a low risk of having a genetic disease.
  • a processing step may be included: processing a biological sample using a DNA methylation status indicator.
  • the genetic region corresponding to the genetic disease is selected from the region encoding the ⁇ -globin gene; preferably, the region encoding the ⁇ -globin gene is selected from The region between 190000-250000 base pairs of human chromosome 16; more preferably, the region encoding the ⁇ -globin gene is selected from any one of the base pairs of human chromosome 16 in the following positions: 202146, 202148 ,202161,202170,202178,202310,202425,205180,205229, 205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284 ,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,2107
  • the region encoding the ⁇ -globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484,229499,229527 , 229535 or any combination of two or more of the above base pairs; preferably, the region encoding the ⁇ -globin gene contains at least the following positions selected from human chromosome 16 Any base pair: 229484, 229499 or a combination of the above base pairs.
  • the DNA methylation status display agent is selected from an antibody or binding protein that recognizes methylated DNA, bisulfite, and an enzyme that has catalytic oxidation of DNA , Enzymes with DNA deamination or methylation-sensitive enzymes, or a combination thereof; preferably, the methylation-sensitive enzyme is selected from methylation-sensitive restriction enzymes, more preferably, the The methylation-sensitive restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
  • reagent required for enriching the characteristic free DNA is selected from the reagents required for the liquid phase hybridization probe capture method and the polymerase chain reaction amplification method.
  • a detection system for detecting whether a fetus has a genetic disease or a risk of having a genetic disease includes the following modules:
  • Detection module the detection module detects the DNA methylation pattern of the total DNA fragments in the processed biological sample; wherein, the biological sample is a plasma sample of a pregnant female;
  • the differentiation module distinguishes different patterns of DNA methylation of the total DNA fragments in the biological sample obtained in the detection step;
  • the calculation module calculates the proportion of DNA fragments carrying a specific DNA methylation pattern in the biological sample to the total DNA fragments in the region, and finds DNA that exists only in placental tissue but not in the plasma of infertile women Methylation mode, that is, fetal-specific DNA methylation mode;
  • the statistic module counts the proportion of fetal-derived DNA fragments of different maternal plasma DNA samples in different gene regions to the total DNA fragments in the region based on the found fetal-specific DNA methylation patterns;
  • Comparison module compares the ratio of the DNA fragments of the fetus in the gene region corresponding to the genetic disease to the DNA fragments of the fetus in the genetic region corresponding to the genetic disease and the DNA from the fetus in the control gene region The proportion of fragments in the total number of DNA fragments in the region;
  • the system may further include a judgment module:
  • the judgment module makes the following judgments:
  • the fetus has a genetic disease or Have a high risk of suffering from genetic diseases;
  • the fetus does not have genetic
  • the disease may or may not have a risk of having a genetic disease or have a low risk of having a genetic disease.
  • the detection system further comprises a processing module: the processing module processes a biological sample using a DNA methylation status indicator.
  • the genetic region corresponding to the genetic disease is selected from the region encoding the ⁇ -globin gene; preferably, the region encoding the ⁇ -globin gene is selected From the region between 190000-250000 base pairs of human chromosome 16; more preferably, the region encoding the ⁇ -globin gene is selected from any one of the base pairs of human chromosome 16 in the following position: 202146, 202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278, 210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,
  • the region encoding the ⁇ -globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484,229499, 229527,229535 or any combination of two or more of the above base pairs; preferably, the region encoding the ⁇ -globin gene contains at least the following positions selected from human chromosome 16 Any one of the base pairs: 229484, 229499 or a combination of the above base pairs.
  • the sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid or cord blood, or Its combination.
  • the DNA methylation status display agent is selected from an antibody or a binding protein that recognizes methylated DNA, bisulfite, and has a catalytic oxidation of DNA Enzymes, enzymes with DNA deamination or methylation sensitive enzymes, or a combination thereof; preferably, the methylation sensitive enzyme is selected from methylation sensitive restriction enzymes, more preferably, The methylation-sensitive restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
  • reagent required for enriching characteristic free DNA is selected from reagents required for liquid phase hybridization probe capture method, polymerase chain reaction amplification method
  • a detection device for detecting whether a fetus has a genetic disease or a risk of genetic disease including:
  • Memory for storing processors and executing instructions
  • the processor is configured to implement the method according to any one of the foregoing 23-31 when executing the processor executable instructions.
  • An aspect of the present disclosure provides a method for detecting whether a fetus has a genetic disease or a risk of suffering from a genetic disease, the method comprising the following steps: detecting DNA methylation of total DNA fragments in a treated biological sample Pattern; distinguish the different patterns of DNA methylation of the total DNA fragments in the biological sample obtained in the detection step; calculate the proportion of DNA fragments carrying a specific DNA methylation pattern in the biological sample to the total DNA fragments in the region, Find the DNA methylation pattern that exists only in placental tissue but not in the plasma of infertile women, that is, fetal-specific DNA methylation pattern; according to the found fetal-specific DNA methylation pattern, count the plasma DNA samples of pregnant women In different gene regions, the proportion of fetal-derived DNA fragments in the total DNA fragments of the region; comparing the ratio of maternal plasma DNA samples to the fetal-derived DNA fragments in the genetic region corresponding to the genetic disease to the total number of DNA fragments in the region The proportion of feta
  • a method of detecting whether a fetus has or is at risk of suffering from ⁇ -thalassemia includes the following steps: detecting the methylation pattern of DNA in a biological sample; distinguishing different patterns of DNA methylation of the total DNA fragments in the biological sample obtained in the detection step; calculating the placental tissue within the ⁇ -globin gene region Compared with the methylation pattern of DNA specific to non-pregnant women's plasma; the number of DNA fragments with fetal-specific methylation pattern in the ⁇ -globin gene region and the control region of pregnant women's plasma DNA accounted for the total number of DNA fragments in this region The ratio of ⁇ -globin gene region and the control region of the DNA fragments with fetal-specific methylation pattern to the total number of DNA fragments in this region.
  • whether the fetus has ⁇ -thalassemia is determined by judging the similarities and differences of the aforementioned ratios.
  • the test sample may be a pregnant woman's peripheral blood, urine, saliva, sweat.
  • the test sample is the peripheral blood of the pregnant woman.
  • the peripheral blood samples of pregnant women are processed to obtain plasma or serum.
  • the plasma sample is tested. After testing the samples of pregnant women's peripheral blood, urine, saliva, sweat, etc., assessing the genotype of the fetal ⁇ -globin gene is a non-invasive detection method.
  • the test sample may be fetal tissue, such as placental tissue, and detecting the sample is an invasive detection scheme.
  • the sample it is not necessary to treat the sample with a DNA methylation status indicator.
  • the sample is treated with a DNA methylation status indicator.
  • the DNA methylation status indicator is used to process the sample, and methylated and non-methylated modified DNA will show differences.
  • the DNA methylation status indicator can be bisulfite, methylation-sensitive restriction enzymes (such as HpaII and BstUI), antibodies or binding proteins that recognize methylated DNA.
  • the sample is processed using bisulfite.
  • the detection method of the DNA methylation pattern used in the step of detecting the methylation pattern of DNA in the sample is a sequencing method.
  • the sample DNA is constructed as a whole genome bisulfite sequencing library, and then the library is sequenced using a detection platform based on the sequencing-by-synthesis principle, such as a next-generation sequencer.
  • the detection method used in step (2) also includes real-time PCR, digital PCR, mass spectrometry, single molecule sequencing, and the like.
  • the number of DNAs with fetal-specific methylation patterns is counted.
  • the methylation pattern of the sample DNA obtained in the step of detecting the methylation pattern of DNA in the sample is counted, and the methylation pattern is used to classify the DNA fragments.
  • this statistical analysis can be implemented using chain analysis, one-to-one correspondence analysis, and so on.
  • machine learning methods can be used to distinguish methylation patterns on free DNA fragments in the detection sample.
  • a DNA fragment can be defined mathematically as a DNA fragment of fetal origin, when the probability of it from the fetus is significantly higher than that from the mother.
  • this statistical analysis can be implemented using techniques well known in the statistical field such as chi-square test, rank sum test, McDonald's test, hypergeometric test, and general t test.
  • a biological sample can be used as a control for comparison, for example, a DNA fragment that is aligned to the genome of the ⁇ -globin gene region and carries a specific methylation pattern can only be obtained from a It was observed in free DNA samples from pregnant women's peripheral blood and could not be observed from a free blood sample from pregnant women carrying homozygous ⁇ -globin gene region deletion mutant fetuses, so the methylation pattern can only be derived from the fetus The ⁇ -globin gene region of the genome. As the applicants in the field are familiar with, such biological samples can be selected in multiple ways.
  • each DNA fragment can be uniquely defined as "a DNA fragment of fetal origin" or "not necessarily a DNA fragment of fetal origin”.
  • the DNA in the ⁇ -globin gene region and the control region (hereinafter collectively referred to as “genomic regions”) is compared. Specifically, the ratio of fetal DNA fragments to the total number of DNA fragments is analyzed. According to statistical principles, this ratio should be consistent between different genomic regions. In a genomic region, the ratio deviates from the average value, which may represent a copy number change in the region of the fetal genome.
  • genetic mutations and genetic defects carried by the fetus may cause changes in the methylation pattern of other genomic regions (hereinafter "associated regions"). By comparing changes in methylation patterns in related regions, it is also possible to speculate on genetic mutations carried by the fetus. In some embodiments, the methylation pattern of the associated region can be altered to speculate whether the fetus carries the ⁇ -globin gene defect.
  • the method is suitable for detecting the diagnosis of fetal ⁇ -thalassemia, which includes ⁇ -globin gene deletion type and mutant type.
  • the method is used to diagnose a fetus with a complete deletion of the ⁇ -globin gene.
  • the fetal ⁇ -globin gene is a homozygote (SEA/SEA) formed by SEA deletion alleles. Fetuses with all alpha-globin genes deleted show anemia with systemic edema, which is prone to intrauterine death and can cause serious complications in pregnant women.
  • the fetal ⁇ -globin gene is a genotype formed by a combination of SEA, THAL, ⁇ 3.7, and ⁇ 4.2 deletion alleles, such as SEA/SEA, SEA/ ⁇ 3.7, SEA/ ⁇ 4.2 etc.
  • the fetal ⁇ -globin gene carries point mutations, such as ⁇ CS, ⁇ WS.
  • the method can also assess the presence or risk of pregnancy-related disorders. For example, the pregnancy-related disorder is premature delivery.
  • a kit for detecting whether a fetus has ⁇ -thalassemia contains the reagents needed to enrich the characteristic free DNA.
  • the kit may further comprise a DNA methylation status indicator.
  • characteristic free DNA is enriched by liquid phase hybridization probe capture.
  • characteristic source free DNA is enriched by polymerase chain reaction amplification (PCR).
  • PCR polymerase chain reaction amplification
  • the characteristic source free DNA is enriched by anchor PCR.
  • the kit includes reagents that use the enriched characteristic free DNA for sequencing-by-synthesis detection (so-called library building reagents).
  • the kit includes reagents that use free DNA for single molecule sequencing detection (i.e., so-called library-building reagents).
  • software for statistical analysis of characteristic free DNA is also provided to compare the fraction of fetal DNA in the ⁇ -globin gene region with a standard control to determine the genotype of the fetal ⁇ -globin gene locus.
  • the present disclosure finds that fetal DNA has a specific methylation pattern in the genomic region where the globin gene is located (for example, the region between 190000-250000 base pairs of human chromosome 16).
  • the methylation pattern is different from that of the maternal DNA.
  • the present disclosure provides many new, highly specific DNA methylation markers for the fetus.
  • the marker can distinguish maternal DNA and fetal DNA at a single molecule level, thereby providing a method for independent analysis of fetal-derived DNA fragments. Therefore, the present disclosure further provides a more accurate, more efficient, and more convenient method for non-invasive diagnosis of fetal ⁇ -thalassemia. The method has shown a high degree of accuracy in the experiments provided by the present disclosure.
  • the present disclosure also provides a kit for prenatal diagnosis of fetal ⁇ -thalassemia.
  • the fetal-specific methylation markers provided by the present disclosure can also be used to determine the presence and content of fetal DNA in maternal free DNA, and further used for other fetal disorders and pregnancy-related disorders of pregnant women or Assessment of disease risk.
  • FIGS 1A-1C show the differential methylation sites between the fetus and the mother found through whole-genome methylation sequencing.
  • FIG. 1A shows that there is a stable level of methylation (black: high; white: low) between each placenta sample and the maternal sample.
  • FIG. 1B shows the aforementioned differential methylation sites of fetus and mother, which are present on each chromosome.
  • FIG. 1C shows the continuous multiple CpG sites distributed in the same genomic region among the aforementioned fetal and maternal differential methylation sites.
  • Figure 2 shows the single-molecule methylation markers of fetal and maternal differences discovered through whole-genome methylation sequencing.
  • part A in Figure 2 shows that at this exemplary genomic location, there is a stable level of methylation (black: high; white: low) at each CpG site between the placenta sample and the maternal sample Differences, at the same time, the placenta-specific single-molecule methylation marker (molecule with white color block) and the maternal-specific single-molecule methylation marker (molecule with black color block) can be observed.
  • Part B in Figure 2 shows that at this exemplary genomic location, there is not necessarily a stable level of methylation (black: high; white: low) at each CpG site between the placenta sample and the maternal sample Differences, but single-molecule methylation markers specific to the placenta (molecules with white patches) are still observed
  • Figure 3 shows fetal-specific methylation markers found in the ⁇ -globin genomic region through whole-genome methylation sequencing.
  • Part A in FIG. 3 shows that there are specific single-molecule methylation markers (black: high; white: low) differences between the placenta sample and the maternal sample. The entire DNA molecule is all demethylated DNA and can only be found in placental samples.
  • Part B in Figure 3 shows the aforementioned specific single-molecule methylation markers, which can only be observed in the villi where the ⁇ -globin genomic region is wild-type or heterozygous carrying the SEA-deleted genotype. SEA-deficient genotype villi and infertile women's peripheral blood cannot be observed.
  • the single-molecule methylation marker can only come from the fetal genome, but not from the maternal genome. It is a specific DNA fragment marker of fetal origin in the ⁇ -globin genome region.
  • this family of single-molecule methylation markers is located in the region of 190000-230000 base pairs (GRCh37 version genome) of human chromosome 16. More specifically, the single-molecule methylation markers of this family include CpG sites and methylation status combinations in Table 6.
  • Figure 4 shows that in the plasma of 68 pregnant women carrying pregnant women with homozygous SEA-deficient genotypes, their normalized ⁇ -globin genomic region has a significantly lower local fetal proportion than other pregnant women. According to the normalized proportion of local fetus after 0.01, the sensitivity is 100% and the specificity is 96%.
  • Figure 5 shows the results of testing the plasma DNA of pregnant women with unknown fetal genotype using the specific methylation pattern of the placenta.
  • the words “include”, “have”, “include” or “include” mean inclusive or open-ended and do not exclude additional, unquoted elements or methods step.
  • “comprising”, “having”, “including” or “containing” may also mean closed, excluding additional, unquoted elements or method steps.
  • 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 usually refers to the methylation of the 5'carbon of the nucleotide cytosine residue. 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 a phosphate group. DNA methylation can also occur in cytosines of nucleotide sequences such as CHG and CHH, where H is adenine, cytosine or thymine. DNA methylation can also occur on non-cytosine, such as N6-methyladenine. In addition, DNA methylation can also be in the form of 5-hydroxymethylcytosine.
  • Methods refers to a single or multiple base positions where methylation modification may occur. For example, CpG site, CHG site or CHH site. In some cases, the methylation site is equivalent to the CpG site.
  • Methods is a description of the overall methylation status of the sample DNA in the region. It can be “methylation state”, “methylation level”, “methylation density” or “single molecule methylation state” and combinations thereof.
  • Methods refers to a feature of a particular genomic locus of a DNA segment that is associated with methylation. Such characteristics include, but are not limited to, whether any cytosine (C) residues in the DNA sequence are methylated, the position of the methylated C residue, and the percentage of methylated C at any particular extension of the residue, And allelic differences due to, for example, differences in allelic origin.
  • methylation mode or “methylation status” also refers to the relative or absolute concentration of methylated C or unmethylated C of any particular residue extension in a biological sample.
  • cytosine (C) residues in the DNA sequence are methylated, it can be called “hypermethylation”; if one or more cytosine (C) residues in the DNA sequence are not methylated , Can be called “low methylation”.
  • one or more cytosine (C) residues within a DNA sequence eg, fetal nucleic acid
  • another sequence eg, relative to maternal nucleic acid
  • cytosine (C) residues within the DNA sequence are not methylated with respect to another sequence from a different source or different individual (eg, mother), then the sequence is viewed relative to the other sequence For low methylation.
  • C cytosine
  • Methods refers to the number of methylated DNA molecules at the methylation site divided by the number of total DNA molecules containing the site to describe a methyl group in the DNA sample The overall methylation status of the chemical site.
  • Methods of “Methylation density” is the number of sites in a region that exhibit methylation divided by the total number of readings covering the sites in the region.
  • Single-molecule methylation state refers to a combination of methylation states of all base sites that can be methylated by a single DNA molecule.
  • the single molecule methylation state may include one, two or more methylation sites.
  • a DNA molecule with two methylation sites may have four single-molecule methylation states, including +-, -+, --, and ++ ("+" indicates that the methylation site is methylated Modified, "-" indicates that the methylation site is not modified by methylation).
  • single molecule methylation status is used to distinguish maternal and fetal DNA in plasma samples of pregnant women.
  • Single molecule methylation state may include at least 1, at least two, at least 3, at least 4, at least 5, at least 6, at least 10, at least 20, at least 40, at least 50, The methylation status of at least 100, at least 300, at least 400, at least 500 or more, or any range of the methylation sites of the above values.
  • DNA methylation status indicator refers to an agent capable of differentially modifying methylated and unmethylated DNA in the sample.
  • Detection marker refers to a biochemical indicator that can mark changes or possible changes in the structure or function of systems, organs, tissues, cells and sub-cells.
  • the detection marker is a single-molecule methylation state of DNA.
  • sample refers to any sample that comes from the human body and contains DNA molecules.
  • the “sample” used for fetal detection may be a peripheral blood sample, a placenta sample, a placental villi sample, an amniotic fluid puncture sample, a cord blood sample, a urine sample, a saliva sample, a sweat sample, and the like.
  • gestational age refers to the embryo after 8 weeks of gestational age.
  • gestational age is a measure of gestational age, where the starting point is the corresponding age estimated by the woman's last normal menstrual period (LMP) or other methods.
  • LMP menstrual period
  • gestational age refers to the age of the embryo or fetus plus two weeks. In humans, although delivery is common within 37 to 42 weeks, delivery usually occurs around 40 weeks of gestational age.
  • “Local fetal proportion” refers to the proportion of fetal DNA fragments to all DNA fragments on the detection area.
  • Fetal-specific methylation pattern refers to the DNA methylation pattern that exists only in placental tissue but not in the plasma of infertile women.
  • ⁇ -thalassemia refers to an inherited hemolytic anemia caused by defects in the ⁇ -globin gene.
  • the genetic defect of ⁇ -thalassemia is mainly caused by the deletion of ⁇ -globin gene.
  • the ⁇ -globin gene cluster is located on chromosome 16 and includes two highly homologous ⁇ -globin genes, ⁇ 1 and ⁇ 2, both of which can express ⁇ -globin chains. If the ⁇ -globin gene on a chromosome is normal, it is recorded as ( ⁇ /), then the genotype of a normal person is ( ⁇ / ⁇ ). The deletion of an ⁇ -gene on a chromosome is recorded as (- ⁇ /), for example (- ⁇ 3.7 /), (- ⁇ 4.2 /), etc.
  • the ⁇ -globin chain cannot be synthesized, which is called ⁇ 0 -thalassemia, and is denoted as (--/), such as (-- SEA /), (-- THAI /) and (- -FIL /), etc.
  • the homozygote shows severe ⁇ -thalassemia, and the affected fetus is prone to intrauterine death.
  • Non-deleted ⁇ -thalassemia generally does not cause two ⁇ -genes on a chromosome to be damaged at the same time, so it is called ⁇ + -thalassemia, and its allele is recorded as ( ⁇ M ⁇ /) or ( ⁇ M / ), such as ( ⁇ WS ⁇ /), ( ⁇ CS ⁇ /), ( ⁇ QS ⁇ /) common in China.
  • WGBS Whole-genome bisulfite sequencing
  • methylated DNA immunoprecipitation sequencing (MeDIP-Seq, Jacinto et al., 2008 44(1), 35–43), to simplify representative sulfurous acid Hydrogen salt sequencing (RRBS, Meissner et al., Nucleic Acids Res, 2005; 33:5868–5877.), methylated microarrays (for example, Infinium Human Methylation 450K BeadChip, Sandoval et al., Epigenetics, 2011, 6 (6), 692–702) and genome-wide bisulfite sequencing (WGBS, Lister et al., Cell, 2008; 133:523–36.), etc.
  • RRBS sulfurous acid Hydrogen salt sequencing
  • WGBS genome-wide bisulfite sequencing
  • the whole genome bisulfite sequencing method can analyze all methylation sites on the genome, and can detect a single DNA molecule at a single base resolution condition. Therefore, the method can comprehensively study the DNA methylation status within the whole genome and provide a quantitative analysis method for the DNA methylation status. Further, the method can be used for single molecule methylation status assessment.
  • the whole-genome bisulfite sequencing method is used to detect the methylation pattern of placental tissue DNA and non-pregnant female plasma free DNA.
  • the placental tissue DNA has a specific single molecule methylation state.
  • the methylation level of a single base site has extremely high noise and is not suitable for single molecule detection.
  • the DNA derived from placental tissue is very different from the single-molecule methylation pattern of plasma-free DNA in non-pregnant women.
  • a whole genome bisulfite sequencing method genotype ⁇ / ⁇ , - SEA / ⁇ and - SEA / - SEA placental tissue after DNA sequencing of the Within the ⁇ -globin gene region, the specific single molecule methylation status of the fetus is analyzed.
  • the genotype ⁇ / ⁇ , - placental DNA SEA / ⁇ , said monomolecular specific DNA methylation status can be detected, while in the absence of alpha] globin gene ( - SEA /-- SEA genotype), the DNA of the specific single molecule methylation state cannot be detected in the DNA of the placenta tissue of the genotype. Therefore, the DNA fragments of the specific single molecule methylation status can be used to determine the specific source of placental tissue.
  • the present disclosure provides a fetal-specific DNA methylation marker that can be used for prenatal diagnosis of fetal ⁇ -thalassemia.
  • the marker is selected from single-molecule methylation markers between 190000-230000 base pairs of human chromosome 16.
  • the marker is a methylation level of base pairs selected from any one of the following positions on human chromosome 16: 202146, 202148, 202161, 202170 ,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320 ,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185 ,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,
  • the marker is a methylation level of base pairs selected from any one of the following positions on human chromosome 16: 229484,229499,229527,229535 (GRch37 version genome) or any combination of two or more of the above base pairs.
  • the marker is a methylation level of base pairs selected from any one of the base pairs of human chromosome 16 in the following positions: 229484, 229499, (GRch37 version Genome) or a combination of the above base pairs.
  • the marker is a methylation level of base pairs selected from any one of the following positions in human chromosome 16 at the following positions: 229527,229535 (GRch37 version genome ) Or a combination of the above base pairs.
  • the base pair is selected from any one of the following positions on human chromosome 16: 229484, 229499, 229527, 229535 (GRch37 version genome) or any two of the above base pairs Or a combination of more than any two base pairs and base pairs selected from one or more of the following positions on human chromosome 16: 202146, 202148, 202161, 202170, 202178, 202310, 202425, 205180, 205229, 205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575, 210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,
  • the present disclosure provides a non-invasive method for prenatal diagnosis of fetal ⁇ -thalassemia, which uses fetal-specific single-molecule methylation status to distinguish fetal DNA from maternal blood in maternal DNA, and then to the fetus The copy number of DNA was counted to assess the genotype of the fetal ⁇ -globin gene.
  • the non-invasive prenatal detection method for ⁇ -thalassemia may include the steps of: detecting the methylation pattern of DNA in the biological sample; distinguishing the DNA of the total DNA fragments in the biological sample obtained in the detection step Different patterns of methylation; calculated methylation patterns of placental tissue within the ⁇ -globin gene region compared to DNA specific to the plasma of non-pregnant women; statistics of maternal plasma DNA in the ⁇ -globin gene region and control region The ratio of the number of DNA fragments in fetal-specific methylation patterns to the total number of DNA fragments in this region; compare the number of DNA fragments with fetal-specific methylation patterns in the ⁇ -globin gene region and the control region to the total DNA fragments in this region The ratio of the number.
  • whether the fetus has ⁇ -thalassemia is determined by judging the similarities and differences of the aforementioned ratios.
  • the non-invasive prenatal detection method of ⁇ -thalassemia involved in the present disclosure may include the steps of: extraction of free DNA from peripheral blood of pregnant women; treatment of samples using DNA methylation status indicator; detection of post-treatment The methylation pattern of DNA in the sample; count the number of DNAs with fetal-specific methylation patterns; analyze the DNA in the ⁇ -globin gene region and the control region compared in the previous statistical step; convert the ⁇ -globin gene region The fetal DNA fraction is compared with the standard control to determine the genotype of the fetal ⁇ -globin gene.
  • Methods for extracting DNA from plasma are well known to those skilled in the art. It can be carried out in accordance with conventional methods for DNA preparation.
  • the DNA in the plasma sample is extracted using Qiagen column extraction kit.
  • the DNA in the plasma sample may not be extracted, for example, a bisulfite salt is used to directly convert the serum sample, and then the DNA methylation status is detected.
  • DNA methylation status display agents are preferred, such as bisulfite.
  • Bisulfite can convert unmethylated cytosine to uracil, while methylated cytosine remains unchanged, and then use polymerase chain reaction to amplify the converted DNA, and uracil to thymus Pyrimidine (Frommer et al. Proc Natl Acad Sci USA, 1992, 89: 1827-31), and then detect whether cytosine is converted into thymine to determine whether the cytosine is methylated.
  • the DNA methylation state display agent also includes methylation-sensitive endonucleases, antibodies or binding proteins that recognize the DNA methylation state, enzymes with DNA oxidant deamination, and the like.
  • the sample may not be treated with a DNA methylation status indicator, for example, a single-molecule sequencing system based on nanopores or a zero-mode waveguide may be used to directly detect the methylation status of DNA molecules.
  • the above methods can be used for prenatal diagnosis of fetal ⁇ -thalassemia.
  • This disclosure can be implemented based on high-throughput sequencing platforms, such as sequencing platforms such as Miseq, Hiseq, Nextseq, and Novaseq of Illumin, sequencing platforms such as Ion Torrent or Ion Proton of Life Technologies, and PacBio RS II of Pacific Biosciences system, etc. Platform, Oxford, Nanopore Technologies GridIONX5 and other sequencing platforms.
  • sequencing platforms such as Miseq, Hiseq, Nextseq, and Novaseq of Illumin
  • sequencing platforms such as Ion Torrent or Ion Proton of Life Technologies
  • PacBio RS II of Pacific Biosciences system
  • a methylation detection method based on the principle of bisulfite conversion has become a conventional technique for detecting DNA methylation status, and is also well known to those skilled in the art.
  • EZ DNA DNA-Methylation-Gold Bisulfite Conversion Kit from Zymo Research is used to perform bisulfite conversion on the sample DNA.
  • the maternal plasma DNA is repaired, added with A, and then ligated with the Illumina methylated adaptor, and then the bisulfite conversion is performed on the ligation product of the DNA and the adaptor and then utilized Polymerase chain reaction (PCR) was used to amplify to obtain a sequencing library, and the sequencing library was sequenced on an Illumina Nextseq 500 sequencer to obtain a genome-wide detection result of the maternal plasma DNA.
  • PCR Polymerase chain reaction
  • maternal plasma DNA is first treated with bisulfite and then a sequencing library is constructed, and then detected using a high-throughput sequencing platform.
  • a variety of currently disclosed methods and commercial kits can construct sequencing libraries for bisulfite-converted DNA samples, for example, random primer-based amplification methods (Fumihito et al., Nucleic Acids Research, 2012, 40( 17): e136-e136; Khanna et al., Nature Methods, 2013, 10(10)), a method based on the connection of single-stranded nucleotides and adaptors (Raine et al., Nucleic Acids Research, 2016, 45(6 ): e36; Gansauge et al., Nucleic Acids Research, 2017, 45(10): e79), a method based on terminal deoxynucleotidyl transferase (TdT) (Peng et al., Nucleic Acids Research, 2015 , 43(6): e35) etc.
  • a sequencing kit is used to construct a sequencing library for the bisulfite-converted DNA sample.
  • the commercial kit may be Illumina's Truseq DNA methylation kit, Swift Biosciences' Accel-NGS Methyl -Seq DNA Library Kit etc.
  • a liquid phase or solid phase probe is used to hybridize and capture the relevant regions, followed by sequencing to complete the targeted detection of the DNA methylation status of the target region.
  • the probe may be complementary to the genomic sequence, for example, the sample DNA is repaired, A is added, and then the Illumina methylated adaptor is ligated, and then the ligation product of the DNA and the adaptor is connected to the The probe is incubated for hybridization, the probe is washed, bisulfite conversion is performed, and then amplified by polymerase chain reaction (PCR) to obtain a sequencing library, and the sequencing library is sequenced on an Illumina sequencer to obtain the sample Detection results of DNA methylation status within the target area (Hing B, Ramos et al., Epigenetics 2015; 10:581-96).
  • the probe may be complementary paired with the bisulfite converted DNA sequence, for example, the sample DNA is repaired, A is added, and then connected to the Illumina methylated adaptor, followed by bisulfite Salt transformation, and then incubating and hybridizing the above amplification product with the probe, washing the probe and performing amplification using polymerase chain reaction (PCR) to obtain a sequencing library, and sequencing the sequencing library on an Illumina sequencer, Obtain the detection results of the methylation status of the sample DNA within the target area (Allum et al., Nat Commun, 2015; 6:7211; Li et al., Nucleic Acids Res. 2015; 43:e81).
  • PCR polymerase chain reaction
  • the probe may be solid phase (Okou et al., Nature Methods, 2007, 4(11): 907; Sandoval et al., Epigenetics, 2011, 6(6), 692-702).
  • the DNA that hybridizes to the probe may be already ligated to the adaptor (Wang et al., Bmc Genomics, 2011, 12(1):597).
  • the DNA that hybridizes to the probe may not be linked to the adaptor.
  • the DNA is hybridized and incubated with an RNA probe, and then the probe is washed, then the RNA probe is degraded using RNase, and then the captured DNA is subjected to bisulfite treatment after library construction, followed by PCR amplification (Or perform bisulfite treatment on the captured DNA and then construct the library, followed by PCR amplification), and finally sequence the sequencing library.
  • the DNA methylation status is detected based on a simplified representative bisulfite sequencing method (RRBS, Meissner et al., Nucleic Acids Res, 2005; 33:5868-5877; Gu et al., Nat Protoc. 2011; 6, (4), 468-481).
  • RRBS Meissner et al., Nucleic Acids Res, 2005; 33:5868-5877; Gu et al., Nat Protoc. 2011; 6, (4), 468-481).
  • the endonuclease used for RRBS sequencing can be selected based on the fetal-specific DNA methylation marker regions provided by the present disclosure.
  • the DNA methylation status is detected by immunoprecipitation sequencing based on methylated DNA antibodies or binding proteins (MeDIP-Seq, Jacinto et al., Biotechniques, 2008; 44(1), 35- 43; MBD-seq, Serre et al., Nucleic Acids Res 2010; 38:391–399.).
  • the DNA methylation status is detected based on methylation-sensitive restriction enzyme sequencing (MRE-seq, Maunakea et al., Nature. 2010; 466:253-257), Methylation-sensitive restriction enzymes can be selected based on the fetal-specific DNA methylation marker regions provided by the present disclosure.
  • the target region is amplified by polymerase chain reaction (PCR), and then the amplified product is sequenced to analyze the methylation status of the sample DNA.
  • PCR polymerase chain reaction
  • the PCR may be multiplex PCR.
  • the PCR amplification is performed using microdrop technology (Komori et al., Genome Research, 2011, 21(10): 1738-1745).
  • Single-molecule sequencing platforms e.g., Oxford Nanopore’s MinION, Pacific Biosciences system’s PacBio RS II and other sequencing platforms
  • Single-molecule sequencing platforms will allow direct detection of the methylation status of DNA molecules (including N6-methyl) without bisulfite conversion Adenine, 5-methylcytosine and 5-hydroxymethylcytosine (Flusberg et al., 7:461-465 Nature methods; J Shim 2013 Sci Rep 3:1389)).
  • the methylation status of sample DNA analyzed using a single molecule sequencing platform without bisulfite conversion.
  • 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 analysis (Plongthongkum et al., Nature Reviews Genetics, 2014, 15(10) :647-61; Consortium et al., Nature Biotechnology, 2016, 34(7):726).
  • mass spectrometry analysis Plongthongkum et al., Nature Reviews Genetics, 2014, 15(10) :647-61; Consortium et al., Nature Biotechnology, 2016, 34(7):726).
  • any method that can detect the DNA methylation status can achieve prenatal diagnosis of fetal ⁇ -thalassemia, including but Not limited to non-invasive prenatal diagnosis of the fetus.
  • Methods is a description of the overall methylation status of the sample DNA in the region.
  • the analysis of DNA methylation patterns may be selected from the analysis of DNA methylation status.
  • the "analysis of DNA methylation patterns" is to count the number of DNAs with fetal-specific methylation patterns.
  • the DNA fragment information obtained by library construction and sequencing can use methods well known to those in the field, such as bismark (Felix Kruegerand Simon R. Andrews (2011). Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications.Bioinformatics), bwa-meth (Brent S. Pedersen, et Al. (2014). Fast and accurate alignment of long bisulfite-seq reads. Bioinformatics) and other methods to convert DNA fragments Compare to the human reference genome, such as: GRCh37 (Feb.2009, hg19).
  • DNA fragments that conform to the fetal-specific DNA methylation pattern provided by the present disclosure can be considered as DNA fragments of fetal origin.
  • Those skilled in the art should readily recognize that, based on the fetal-specific DNA methylation markers provided by the present disclosure, using any reasonable statistical means to classify the DNA fragments, the prenatal diagnosis of fetal ⁇ -thalassemia can be achieved.
  • the prenatal diagnosis of fetal ⁇ -thalassemia can be selected from non-invasive prenatal diagnosis of the fetus.
  • the "judgment of the genotype of the fetal ⁇ -globin gene” may be the result of the analysis step of the DNA methylation pattern, such as counting the number of DNAs with fetal specific methylation pattern, and comparing DNA into the ⁇ -globin gene region and the control region. The fetal DNA fraction in the ⁇ -globin gene region is compared with the standard control to determine the genotype of the fetal ⁇ -globin gene.
  • the fetal-derived DNA fragments can be counted on the control genomic region (non- ⁇ -globin genomic region) and ⁇ -globin genomic region number. Furthermore, the ratio of the DNA fragments derived from the fetus to all the DNA fragments in the region in a certain genomic region is calculated (hereinafter referred to as "local fetal proportion").
  • the local fetal proportion is defined by a single molecule methylation status. In other embodiments, the local fetal proportion may be defined by two or more single molecule methylation states.
  • the local fetal source ratio is the average of the single-molecule methylation state corresponding to the fetal source ratio. In other embodiments, the local fetal source ratio is the single-molecule methylation state corresponding to the fetal source ratio. Weighted average. Since the control genome region is always normal by default, the ratio of the local fetal proportion of the ⁇ -globin genomic region and the local fetal proportion of the control genomic region (hereinafter referred to as the relative ⁇ -globin gene ratio) is compared, The copy number of the fetal ⁇ -globin genomic region can be estimated.
  • the target pregnant woman carries the fetal ⁇ -globin genotype.
  • the prenatal diagnosis of fetal ⁇ -thalassemia can be achieved, including but not limited to fetal Non-invasive prenatal diagnosis.
  • the non-invasive prenatal detection method of ⁇ -thalassemia may include the following steps: obtaining a test sample; preprocessing the test sample.
  • the step of "obtaining a test sample” is a step of obtaining peripheral blood of a pregnant woman.
  • the step of obtaining the peripheral blood of a pregnant woman includes the following steps: collecting a biological sample of a pregnant woman containing maternal DNA and fetal DNA, the biological sample including but not limited to the blood, urine and saliva of the pregnant woman, preferably a blood sample of the pregnant woman.
  • the blood sample includes but is not limited to the peripheral blood of pregnant women.
  • Collect peripheral blood of pregnant women suitable for gestational age, the gestational age can be 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 28 weeks , 32 weeks, 36 weeks, 40 weeks, or any time between the above time points, preferably 6 weeks to 28 weeks, more preferably 10 weeks to 22 weeks.
  • the container for collecting peripheral blood of pregnant women can be a commercial product such as Streck Tube blood collection tube.
  • the step of "pretreatment of the test sample” is a step of preparing blood plasma of pregnant women.
  • the preparation steps of the peripheral blood plasma of pregnant women include the following steps: A method of separating plasma or serum from maternal peripheral blood is well known to those skilled in the art.
  • the peripheral blood sample of the pregnant woman is centrifuged at 1600 g to obtain plasma or serum.
  • the peripheral blood sample collected from the Streck Tube blood collection tube is centrifuged at 1600g at 4°C, and then the plasma is transferred to a new centrifuge tube, and the plasma is then subjected to 16000g at 4°C. Centrifuge to remove residual blood cells.
  • Example 1 Methylation status of DNA in placental tissue and free DNA in plasma of infertile women Determination
  • peripheral blood sample was centrifuged at 1600g for 10 minutes at 4°C, and the plasma fraction was centrifuged at 16000g for 10 minutes at 4°C to further remove residual blood cells.
  • DNA was extracted from the placental tissue using the DNeasy Blood & Tissue kit (manufacturer: QIAGEN; catalog number: 69506). According to the manufacturer's instructions, free DNA was extracted from the plasma using QIAamp Circulating Nucleic Acid Extraction Kit (manufacturer: QIAGEN; catalog number: 55114).
  • HyperPreP library construction kit 1 ⁇ g of fragmented placental tissue DNA or 10-50 ng of non-pregnant female free DNA was repaired, A was added, and then an Illumina methylated Truseq adaptor (primer sequence number; 001/002) was connected.
  • the sources of the reagents or kits used in the foregoing steps are shown in Table 2A.
  • the sources of the reagents or kits used in the foregoing steps are shown in Table 2B.
  • the ligation reaction solution was prepared as shown in Table 5, mixed thoroughly, and reacted at 20°C for 15 minutes.
  • Reaction component Volume AgencourtAMPure XP magnetic beads (DNA) - Nuclease-free Water 30 5X KAPALigationBuffer 10 Adaptor (10 ⁇ M, 001/002) 5 KAPADNALigase 5
  • KAPA HiFi Hot Start Uracil + Ready Mix PCR amplification kit (manufacturer: Roche Kapa Biosystems; catalog number: KK2802) was used for PCR amplification of bisulfite-converted DNA. After the PCR reaction was completed, DNA was purified using AgencourtAMPure XP magnetic beads to obtain a sequencing library.
  • Agilent 2100 Bioanalyzer Agilent Technologies, catalog number G2939BA
  • Agilent High Sensitive DNA Kit Agilent Technologies, catalog number 5067-4626
  • Quantitative kits KAPA Biosystems, catalog number KK4824
  • DNA quantification standards and premix primer kits KAPA Biosystems, catalog number KK4808 were used to detect sequencing library concentrations.
  • the sequencing library was sequenced using the IlluminaNextseq sequencer.
  • Example 2 Based on the sequencing results obtained in Example 1, further data analysis was performed. Use bwa-meth(Version:0.2.0) to compare the sequenced data. After the comparison, the single molecule methylation pattern of the DNA fragment was extracted by using the epiread command of the biscuit toolkit (version:0.2.0.20161222). According to the analysis of the statistical software package MASS (version: 7.3.51.1) in R language, a specific single-molecule methylation pattern stably existing in the placental genome compared to the free DNA genome of infertile women was obtained.
  • MASS version: 7.3.51.1
  • Example 3 Methylation of DNA in placental villi and free DNA in plasma of infertile women State determination
  • peripheral blood sample was centrifuged at 1600g for 10 minutes at room temperature, and the plasma fraction was centrifuged at 16000g for 10 minutes at room temperature to further remove residual blood cells. .
  • DNA was extracted from placental villi tissue using DNeasy Blood & Tissue kit (manufacturer: QIAGEN; catalog number: 69506). According to the manufacturer's instructions, free DNA was extracted from the plasma using QIAamp Circulating Nucleic Acid Extraction Kit (manufacturer: QIAGEN; catalog number: 55114).
  • the implementation method is the same as the corresponding part in Example 1.
  • Post-sequencing analysis use bwa-meth(Version:0.2.0) to compare the data after sequencing. According to the fetal-specific single-molecule methylation markers found in Example 2, use samtools (Version: 1.3.1) ⁇ Heng Li Li et al. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics ⁇ to extract all DNA fragments covering fetal-specific methylation markers. Use the epiread command of the biscuit toolkit (Version:0.2.0.20161222) to extract the single-molecule methylation pattern of the above DNA fragment. Through the analysis of the statistical software package MASS (Version: 7.3.51.1) in R language, fragments corresponding to the methylation markers of this model were obtained.
  • Example 4 Analysis of fetal-specific methylation markers in the ⁇ -globin genomic region
  • Table 6 Fetal-specific single-molecule methylation patterns (wherein the left column in Table 6 is “specific single-molecule methylation status" and the right column is "specific genomic location information where single-molecule methylation status is located”)
  • Example 5 Analysis of blood of pregnant women with known fetal genotype using the methylation pattern specific to the placenta Plasma DNA
  • Post-sequencing analysis use bwa-meth(Version:0.2.0) to compare the data after sequencing.
  • samtools (Version: 1.3.1) ⁇ Heng Li Li et al. (2009).
  • the Sequence Alignment/Map format and SAMtools.Bioinformatics ⁇ extract all DNA fragments that are compared to the target area and the control area.
  • Use the epiread command of the biscuit toolkit (Version:0.2.0.20161222) to extract the single-molecule methylation pattern of the above DNA fragment.
  • MASS (Version: 7.3.51.1) in R language, fragments corresponding to the markers of fetal specific methylation pattern were obtained.
  • the local fetal proportion is calculated for the ⁇ -globin genomic region and the control region, and the ⁇ -globin is calculated according to the local fetal proportion in the control region
  • the proportion of local fetal origin in the genomic region is normalized.
  • Example 6 Using the specific methylation pattern of the placenta to test the blood of pregnant women with unknown fetal genotype Plasma DNA
  • the normalized sample with a local fetal ratio of less than 0.01 in the ⁇ -globin genomic region is a pregnant woman who may carry a fetus with a homozygous SEA deletion genotype (ie, a child with severe ⁇ -thalassaemia).
  • Results The experimental results are shown in Table 7 and Figure 5.
  • 50 patients with homozygous SEA-deleted fetuses were correctly identified by amniotic fluid puncture test, with a correct rate of 99% (50/51), and correctly identified the heterozygous SEA-deleted genotype or completely wild-type
  • the sensitivity is 98.04% and the specificity is 98.89%.
  • an undetected child with severe thalassemia was an individual with placenta fitting. Therefore, the method described in this patent can non-invasively detect whether the fetus is pregnant with severe ⁇ -thalassaemia before delivery.

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Abstract

Provided is a use of a testing agent for a fetal-specific DNA methylation pattern in the preparation of an agent or kit for testing a fetus for a genetic disease or the risk of having a genetic disease. Also provided are a method for non-invasive prenatal testing of a fetus for a genetic disease, a testing kit for use with the method, a related testing method and testing system, a readable storage medium corresponding to the testing system, and a testing device. The provided testing kit, the related testing method and testing system, the readable storage medium corresponding to the testing system, and the testing device enable more accurate, more efficient and more convenient non-invasive testing for a fetal genetic disease; especially α-thalassemia.

Description

一种无创产前检测胎儿是否患有遗传疾病的方法A non-invasive method for detecting whether a fetus has a genetic disease before birth 技术领域Technical field
本公开涉及可用于非侵入式产前诊断及筛查的一系列人类基因组位点上的甲基化修饰及其组合。更具体地，本公开涉及一系列具有胎盘组织特异性的人类基因组位点甲基化修饰组合，可用于胎儿是否患有遗传疾病或患有遗传疾病的风险度的非侵入式产前诊断及筛查，例如可用于非入侵式诊断及筛查α地中海贫血病。本公开还涉及基于该系列基因组位点甲基化修饰进行α地中海贫血病非侵入式产前诊断及筛查的实验方法，以及相关的高通量测序试剂盒。The present disclosure relates to a series of methylation modifications and combinations of human genomic loci that can be used for non-invasive prenatal diagnosis and screening. More specifically, the present disclosure relates to a series of combinations of methylation modification of human genomic loci that are specific to placental tissues, and can be used for non-invasive prenatal diagnosis and screening of whether or not the fetus has genetic disease or the risk of genetic disease For example, it can be used for non-invasive diagnosis and screening for alpha thalassemia. The present disclosure also relates to an experimental method for non-invasive prenatal diagnosis and screening of alpha thalassemia based on this series of genomic site methylation modifications, and related high-throughput sequencing kits.
背景技术Background technique
α-地中海贫血(α-Thalassaemia，简称α-地贫)是由于α-珠蛋白基因突变或缺失导致血红蛋白α肽链合成减少或抑制引起的遗传性贫血症。α-地贫多发生于热带和亚热带地区，例如东南亚、印度次大陆和中国南方。在世界范围内，α-地贫是最常见的单基因遗传疾病之一，α-地贫基因的携带率达5％。在中国大陆，α-地贫基因的携带率为7.88％，广西省高达14.13％。Alpha-thalassaemia (α-Thalassaemia, referred to as α-thalassaemia) is an inherited anemia caused by decreased or suppressed synthesis of hemoglobin α peptide chain due to mutation or deletion of α-globin gene. Alpha-thalassaemia occurs mostly in tropical and subtropical regions, such as Southeast Asia, the Indian subcontinent, and southern China. In the world, α-thalassaemia is one of the most common single-gene genetic diseases, and the carrying rate of α-thalassaemia genes is 5%. In mainland China, the carrying rate of α-thalassaemia gene is 7.88%, and it is as high as 14.13% in Guangxi province.
α-地贫的临床表现与α-珠蛋白基因数量直接相关，当α-珠蛋白基因全部缺失(--/--，重型α-地贫)时，患者在胎儿时期发病，表现为重度贫血伴全身水肿等症状(HbBarts'hydropsfetalis，巴氏胎儿水肿综合征)。该疾病对胎儿来说是致命的，同时可能对母体带来严重的甚至危及生命的并发症，如果没有得到足够的医疗护理，孕妇死亡率可能会接近50％。此外，由于α-地贫的流行区域与疟疾流行区域重合，该遗传病特别对欠发达国家和地区造成了沉重的公共卫生负担。The clinical manifestations of α-thalassemia are directly related to the number of α-globin genes. When all α-globin genes are deleted (--/--, severe α-thalassemia), the patient develops fetus during the fetal period, which is manifested as severe anemia Accompanied by systemic edema and other symptoms (HbBarts'hydropsfetalis, Bartholin's fetal edema syndrome). The disease is fatal to the fetus, and may cause serious or even life-threatening complications to the mother. Without adequate medical care, the maternal mortality rate may be close to 50%. In addition, because the endemic areas of α-thalassaemia coincide with the malaria-endemic areas, the genetic disease particularly places a heavy public health burden on less developed countries and regions.
目前，α-地贫的标准产前诊断方法是对高危夫妇进行基因分型筛查，进而采用绒毛活检术、羊膜腔穿刺术等方法对胎盘或胎儿细胞进行产前诊断(Benz EJ Jr.2011.Newborn screening for a-thalassemia keeping up with  globalization.NEngl JMed364:770–771.)。然而，这些常规方法是侵入式的，可能会对胎儿和孕妇造成损伤、甚至有胎儿流产的风险，同时也会增加孕妇的心理负担。有研究显示，绒毛活检术、羊膜腔穿刺术的手术流产风险分别为0.22％、0.11％(Akolekar et al.,Ultrasound ObstetGynecol,2015,45(1):16-26)。At present, the standard prenatal diagnosis method for α-thalassaemia is to perform genotyping screening for high-risk couples, and then to use prenatal diagnosis of placenta or fetal cells using villous biopsy, amniocentesis, etc. (Benz EJ Jr. 2011 .Newborn screening for a-thalassemia keeping up with globalization.NEngl JMed364:770–771.). However, these conventional methods are invasive, may cause damage to the fetus and pregnant women, and even have the risk of fetal abortion, while also increasing the psychological burden of pregnant women. Studies have shown that the risk of surgical abortion in villous biopsy and amniocentesis is 0.22% and 0.11%, respectively (Akolekar et al., Ultrasound Obstet Gynecol, 2015, 45(1): 16-26).
孕妇外周血中胎儿无细胞DNA(Fetal cell-free DNA，cffDNA)的发现使得以cffDNA作为标记物分析胎儿基因状态的非侵入式产前检测(Non-invasive Prenatal Testing，NIPT)成为可能。相较于传统侵入性方法，NIPT具有安全、无并发症、早期诊断等优势，已经被成功应用于胎儿性别鉴定、胎儿RhD血型(rehesus D)基因型鉴定、胎儿染色体非整倍体筛查(例如唐氏综合征、爱德华氏综合征、帕陶氏综合症)。利用cffDNA进行单基因遗传病的无创产前诊断如β-地中海贫血病和威尔逊病等也已有部分科研结果(Chiu et al.,Lancet 360:998-1000,2002；Lv W et al.,ClinChem 61:172–181)。The discovery of fetal cell-free DNA (cffDNA) in the peripheral blood of pregnant women makes it possible to use cffDNA as a marker to analyze non-invasive prenatal testing (NIPT). Compared with traditional invasive methods, NIPT has the advantages of safety, no complications, and early diagnosis. It has been successfully used in fetal sex identification, fetal RhD blood group (rehesus D) genotype identification, and fetal chromosome aneuploidy screening ( (Eg Down syndrome, Edward's syndrome, Patau's syndrome). The use of cffDNA for noninvasive prenatal diagnosis of single-gene genetic diseases such as β-thalassemia and Wilson disease also has some scientific research results (Chiu et al., Lancet 360:998-1000, 2002; Lv W et al., ClinChem 61:172–181).
然而，直至目前，α-地贫的无创诊断面临着巨大的挑战。重型α-地贫(--/--)是由于α-珠蛋白基因的长片段缺失型(例如-- SEA亚型)导致，而无细胞DNA(Cell-free DNA，cfDNA，也称为游离DNA)呈高度片段化状态(Lo,Y.M.D.et al.(2010)Sci.Transl.Med.2,61ra91)，因此，传统的α-地贫基因检测方法，例如缺口聚合酶链式反应(Gap-PCR)、多重连接探针扩增技术(MLPA)等，并不适用于α-地贫基因的无创检测。 However, until now, the non-invasive diagnosis of α-thalassaemia has faced huge challenges. Severe α-thalassaemia (--/--) is caused by the deletion of a long fragment of the α-globin gene (for example, the SEA subtype), and cell-free DNA (cfDNA, also known as free DNA) is highly fragmented (Lo, YMD et al. (2010) Sci. Transl. Med. 2, 61ra91). Therefore, traditional methods for detecting α-thalassaemia genes, such as gap polymerase chain reaction (Gap-PCR ), Multiple Ligation Probe Amplification Technology (MLPA), etc., are not suitable for non-invasive detection of α-thalassaemia genes.
有研究表明，通过检测母体游离DNA中α-珠蛋白基因相关的，父源特异的微卫星序列或单核苷酸多态性位点(SNP)，可以排除部分遗传父方野生型α-珠蛋白等位基因的胎儿，但至少50％的胎儿的基因型不能明确(Ho et al.,(2010)PrenatDiagn.30(1):65-73.；TZ Yan et al.,(2011)PLoS One.6(9):e24779)。基于等位基因剂量，采用实时PCR或高通量靶向测序技术评估母体游离DNA中α-珠蛋白基因的相对含量，可用于判断胎儿α-珠蛋白基因的基因型，但此类方法的准确性依赖于胎儿游离DNA浓度(Long et al.,(2009)ZhonghuaXue Ye XueZaZhi.30(3):175-8.Ge et al.,(2013)PLoS One.2013 Jun  28；8(6):e67464.)。除了上述研究，近年的研究表明，利用父母染色体上α-珠蛋白基因关联的单倍型区域的SNP信息，通过分析母体游离DNA中α-珠蛋白基因的相对单倍型剂量(Relative haplotype dosage，RHDO)能够鉴定胎儿α-珠蛋白基因的基因型(W Wang et al.,(2017)Genet Test Mol Biomarkers.2017 Jul；21(7):433-439.)。然而，尽管单倍体分型技术已经取得了长足进步，基于相对单倍型剂量的无创诊断方法需要事先构建父母双方的单倍体模型，检测成本较高而且周期较长，较难大规模应用。Studies have shown that by detecting the parent-specific microsatellite sequence or single nucleotide polymorphism site (SNP) related to the α-globin gene in free maternal DNA, part of the genetic father's wild-type α-globin can be excluded Fetuses with alleles, but at least 50% of fetuses cannot be genotyped (Ho et al., (2010) Prenat Diagn. 30 (1): 65-73.; TZ Yan Yan et al., (2011) PLoS One. 6(9):e24779). Based on the allele dose, real-time PCR or high-throughput targeted sequencing technology is used to assess the relative content of α-globin gene in maternal free DNA, which can be used to determine the genotype of fetal α-globin gene, but the accuracy of such methods Sex depends on the concentration of fetal free DNA (Long et al., (2009) ZhonghuaXue Ye XueZaZhi.30(3): 175-8. Ge et al., (2013) PLoS One. 2013 Jun 28; 8(6): e67464 .). In addition to the above studies, recent studies have shown that using the SNP information of the haplotype region associated with the α-globin gene on the parent chromosome, by analyzing the relative haplotype dose of the α-globin gene in the maternal free DNA (Relative haplotype dosage, RHDO) can identify the genotype of fetal α-globin gene (Wang Wang et al., (2017) Genet Test Mol Biomarkers. 2017 Jul; 21(7):433-439.). However, although haplotype typing technology has made great progress, non-invasive diagnostic methods based on relative haplotype doses need to construct haploid models of both parents in advance, and the detection cost is higher and the period is longer, which is difficult to apply on a large scale. .
也就是说，不论是基于等位基因平衡还是相对单倍型剂量的检测方法，它们本质上是间接检测胎儿游离DNA，因此，母体游离DNA将会产生较大的背景噪音。在母体血浆中，胎儿DNA不但含量低，而且还与大量母体DNA共同存在(Lo YMD et al.,Am J Hum Genet 1998；62:768–75；Lun,F.M et al.,Clin.Chem.2008,54,1664–1672)。因此，母体DNA背景对检测结果干扰较大。尽管有成功开展胎儿α-地中海贫血的无创产前基因检测的案例，但是样本中胎儿游离DNA浓度较高，中位值为23.41％，远远超过平均水平(Ge et al.,PLoS One.2013；8(6):e67464)。所以，为了避免母体DNA干扰，进而更高效、准确的评估胎儿α-珠蛋白基因的基因型，需要鉴定特异的，并且不依赖胎儿基因组多态性位点或胎儿性别等限制因素的，独立的胎儿DNA标记物。That is to say, whether it is based on allele balance or relative haplotype dose detection methods, they are essentially indirect detection of fetal free DNA, so free maternal DNA will generate greater background noise. In maternal plasma, the content of fetal DNA is not only low, but also coexist with a large amount of maternal DNA (Lo YMD et al., Am J Hum Genet 1998; 62:768–75; Lun, FM et al., Clin. Chem. 2008 ,54,1664–1672). Therefore, the background of maternal DNA greatly interferes with the detection results. Despite successful cases of non-invasive prenatal genetic testing for fetal alpha-thalassemia, the fetal free DNA concentration in the sample is high, with a median value of 23.41%, far exceeding the average level (Ge et al., PLoS One. 2013 ; 8(6):e67464). Therefore, in order to avoid maternal DNA interference and to more efficiently and accurately assess the genotype of the fetal α-globin gene, it is necessary to identify specific, independent of the limiting factors such as polymorphic sites of the fetal genome or fetal gender, independent Fetal DNA markers.
所以，为了避免母体DNA干扰，进而更加准确、简便、经济、高效的评估胎儿α-珠蛋白基因的基因型，进而进行α-地贫无创产前诊断，需要鉴定特异的，并且不依赖胎儿基因组多态性位点或胎儿性别等限制因素的，独立的胎儿DNA标记物。Therefore, in order to avoid maternal DNA interference, and to more accurately, easily, economically, and efficiently assess the genotype of the fetal α-globin gene, and then perform non-invasive prenatal diagnosis of α-thalassaemia, it is necessary to identify specific and not dependent on the fetal genome Polymorphic site or fetal sex and other limiting factors, independent fetal DNA markers.
发明内容Summary of the invention
发明要解决的问题Problems to be solved by the invention
本公开提供了可用作胎儿α-地中海贫血病的无创产前基因诊断的胎儿DNA标记物，检测方法和检测试剂盒。与现有技术相比，本公开涉及的技术方案能够更加安全、高效、准确、经济地对胎儿α-地中海贫血病开展无创产 前基因诊断。The present disclosure provides fetal DNA markers, detection methods, and detection kits that can be used for non-invasive prenatal genetic diagnosis of fetal alpha-thalassemia. Compared with the prior art, the technical solution related to the present disclosure can carry out non-invasive prenatal genetic diagnosis of fetal α-thalassemia more safely, efficiently, accurately and economically.
用于解决问题的方案Solutions for solving problems
本公开包括但不限于涉及的如下技术方案。The present disclosure includes but is not limited to the following technical solutions involved.
1.一种检测胎儿特异性DNA甲基化模式的试剂在制备用于检测胎儿是否患有遗传疾病或患有遗传疾病的风险度的试剂或试剂盒中的用途，所述胎儿特异性DNA甲基化模式为DNA单分子甲基化状态；其中，所述DNA单分子甲基化状态的甲基化位点选自编码α-珠蛋白基因的区域；优选的，所述胎儿遗传疾病为α-地中海贫血。1. Use of a reagent for detecting fetal-specific DNA methylation patterns in the preparation of a reagent or kit for detecting whether a fetus has a genetic disease or a risk of suffering from a genetic disease The methylation mode is a single-molecule DNA methylation state; wherein, the methylation site of the single-molecule DNA methylation state is selected from the region encoding the α-globin gene; preferably, the fetal genetic disease is α -Thalassemia.
2.根据1所述的用途，其中，所述编码α-珠蛋白基因的区域选自人类第16号染色体190000-250000碱基对之间的区域；优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体如下位置中的任意一个碱基对：202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合。2. The use according to 1, wherein the region encoding the α-globin gene is selected from the region between 190000-250000 base pairs of human chromosome 16; preferably, the encoding α-globin gene Is selected from any one of the base pairs of human chromosome 16 at the following positions: 202146, 202148, 202161, 202170, 202178, 202310, 202425, 205180, 205229, 205234, 205245, 209848, 209885, 209922, 209959, 209990 ,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763 ,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665 , 221709, 221740, 221748, 221767, 221800, 221810, 229406, 229454, 229484, 229499, 229527, 229535, or any combination of two or more of the above base pairs.
3.根据1-2任一项所述的用途，其中，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合；优选的，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499或上述碱基对的组合。3. The use according to any one of 1-2, wherein the region encoding the α-globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484, 229499, 229527 , 229535 or any combination of two or more of the above base pairs; preferably, the region encoding the α-globin gene contains at least the following positions selected from human chromosome 16 Any base pair: 229484, 229499 or a combination of the above base pairs.
4.根据1-3任一项所述的用途，其中，所述检测胎儿特异性DNA甲基化 模式的试剂选自DNA甲基化状态显示剂和/或检测来自孕妇的生物样本中胎儿特异性甲基化模式DNA的存在的试剂；优选的，所述检测所述来自孕妇的生物样本中胎儿特异性甲基化模式DNA的存在的试剂选自富集特征游离DNA所需的试剂。4. The use according to any one of 1-3, wherein the reagent for detecting fetal-specific DNA methylation patterns is selected from DNA methylation status indicators and/or for detecting fetal specificity in biological samples from pregnant women A reagent for the presence of sexual methylation pattern DNA; preferably, the reagent for detecting the presence of fetal-specific methylation pattern DNA in the biological sample from a pregnant woman is selected from reagents required for enriching characteristic free DNA.
5.根据1-4任一项所述的用途，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。5. The use according to any one of 1-4, wherein the DNA methylation status display agent is selected from an antibody or a binding protein that recognizes methylated DNA, bisulfite, and an enzyme that has catalytic oxidation of DNA , Enzymes with DNA deamination or methylation-sensitive enzymes, or a combination thereof; preferably, the methylation-sensitive enzyme is selected from methylation-sensitive restriction enzymes, more preferably, the The methylation-sensitive restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
6.根据1-5任一项所述的用途，其中，所述富集特征游离DNA所需的试剂选自液相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。6. The use according to any one of 1-5, wherein the reagent required for enriching characteristic free DNA is selected from reagents required for the liquid-phase hybridization probe capture method and the polymerase chain reaction amplification method. The reagents needed, the reagents required for the anchoring nucleic acid amplification method, the reagents for sequencing-by-synthesis detection, or the reagents for single molecule sequencing, or a combination thereof.
7.根据1-6任一项所述的用途，其中，所述样本选自全血、外周血、血浆、血清、尿液、唾液、汗液，胎盘、胎盘绒毛、羊水或脐血，或其组合。7. The use according to any of 1-6, wherein the sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid or cord blood, or combination.
8.根据1-7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。8. The use according to any one of 1-7, wherein the test is a prenatal test; preferably, the prenatal test is a non-invasive prenatal test.
9.一种试剂盒，其用于检测来自孕妇的生物样本中胎儿特异性DNA甲基化模式的存在或不存在，所述试剂盒包含：9. A kit for detecting the presence or absence of fetal-specific DNA methylation patterns in biological samples from pregnant women, the kit comprising:
(a)检测所述生物样本中胎儿特异性DNA甲基化模式的存在的试剂；(a) reagents for detecting the presence of fetal-specific DNA methylation patterns in the biological sample;
可选的，所述试剂盒还包含：Optionally, the kit further includes:
(b)DNA甲基化状态显示剂；(b) DNA methylation status indicator;
其中，当(a)中的试剂不依赖于(b)中DNA甲基化状态显示剂时，试剂盒中不包含(b)；Where, when the reagent in (a) does not depend on the DNA methylation status indicator in (b), (b) is not included in the kit;
优选的，所述胎儿特异性DNA甲基化模式为胎儿特异性DNA单分子甲基化状态。Preferably, the fetal-specific DNA methylation mode is a single-molecule methylation state of fetal-specific DNA.
10.根据9所述的试剂盒，其用于检测胎儿是否患有遗传疾病或患有遗传 疾病的风险度；优选的，所述遗传疾病为α-地中海贫血。10. The kit according to 9, which is used to detect whether a fetus has a genetic disease or a risk of suffering from a genetic disease; preferably, the genetic disease is α-thalassemia.
11.根据9-10任一项所述的试剂盒，其中，所述胎儿特异性DNA单分子甲基化状态的甲基化位点选自编码α-珠蛋白基因的区域；优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体190000-250000碱基对之间的区域；更优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体如下位置中的任意一个碱基对：202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合。11. The kit according to any one of 9-10, wherein the methylation site of the methylation state of the single molecule of fetal-specific DNA is selected from the region encoding the α-globin gene; preferably, The region encoding the α-globin gene is selected from the region between 190000-250000 base pairs of human chromosome 16; more preferably, the region encoding the α-globin gene is selected from the following positions on human chromosome 16 Any one of the base pairs: 202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068, 210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837, 210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800, 221810, 229406, 229454, 229484, 229499, 229527, 229535, or any combination of two or more of the above base pairs.
12.根据9-11任一项所述的试剂盒，其中，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合；优选的，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499或上述碱基对的组合。12. The kit according to any one of 9-11, wherein the region encoding the α-globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484,229499, 229527,229535 or any combination of two or more of the above base pairs; preferably, the region encoding the α-globin gene contains at least the following positions selected from human chromosome 16 Any one of the base pairs: 229484, 229499 or a combination of the above base pairs.
13.根据9-12任一项所述的试剂盒，其中，所述样本选自全血、外周血、血浆、血清、尿液、唾液、汗液，胎盘、胎盘绒毛、羊水或脐血，或其组合。13. The kit according to any one of 9-12, wherein the sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid or cord blood, or Its combination.
14.根据9-13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。14. The kit according to any one of 9-13, wherein the DNA methylation status indicator is selected from an antibody or a binding protein that recognizes methylated DNA, bisulfite, and has a catalytic oxidation of DNA Enzymes, enzymes with DNA deamination or methylation sensitive enzymes, or a combination thereof; preferably, the methylation sensitive enzyme is selected from methylation sensitive restriction enzymes, more preferably, The methylation-sensitive restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
15.根据9-14任一项所述的试剂盒，其中，所述富集特征游离DNA所需的试剂选自液相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。15. The kit according to any one of 9-14, wherein the reagent required for enriching characteristic free DNA is selected from reagents required for the liquid phase hybridization probe capture method, polymerase chain reaction amplification method The reagents required, the reagents required for the anchored nucleic acid amplification method, the reagents for sequencing-by-synthesis detection, or the reagents for single molecule sequencing, or a combination thereof.
16.一种试剂盒，其用于检测胎儿是否患有遗传疾病或患有遗传疾病的风险度，所述试剂盒包含：16. A kit for detecting whether a fetus has a genetic disease or a risk of suffering from a genetic disease, the kit comprising:
(a)检测所述生物样本中胎儿特异性DNA甲基化模式的存在的试剂；(a) reagents for detecting the presence of fetal-specific DNA methylation patterns in the biological sample;
可选的，所述试剂盒还包含：Optionally, the kit further includes:
(b)DNA甲基化状态显示剂；(b) DNA methylation status indicator;
其中，当(a)中的试剂不依赖于(b)中DNA甲基化状态显示剂时，试剂盒中不包含(b)；Where, when the reagent in (a) does not depend on the DNA methylation status indicator in (b), (b) is not included in the kit;
优选的，所述胎儿特异性DNA甲基化模式为胎儿特异性DNA单分子甲基化状态。Preferably, the fetal-specific DNA methylation mode is a single-molecule methylation state of fetal-specific DNA.
17.根据16所述的试剂盒，其通过检测来自孕妇的生物样本中胎儿特异性DNA甲基化模式，进而检测胎儿是否患有遗传疾病或患有遗传疾病的风险度；优选的，所述遗传疾病为α-地中海贫血。17. The kit according to 16, which detects fetal-specific DNA methylation patterns in biological samples from pregnant women, and thereby detects whether the fetus has a genetic disease or a risk of suffering from a genetic disease; preferably, the The genetic disease is α-thalassemia.
18.根据16-17任一项所述的试剂盒，其中，所述单分子甲基化状态的甲基化位点选自编码α-珠蛋白基因的区域；优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体190000-250000碱基对之间的区域；更优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体如下位置中的任意一个碱基对：202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505, 221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合。18. The kit according to any one of 16-17, wherein the methylation site of the single-molecule methylation state is selected from a region encoding an α-globin gene; preferably, the encoding α-globin The region of the globin gene is selected from the region between 190000-250000 base pairs of human chromosome 16; more preferably, the region encoding the α-globin gene is selected from any one of the following positions on human chromosome 16 Base pair: 202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215, 210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873, 210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505, 221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406, 229454, 229484, 229499, 229527, 229535 or any combination of two or more of the above base pairs.
19.根据16-18任一项所述的试剂盒，其中，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合；优选的，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499或上述碱基对的组合。19. The kit according to any one of 16-18, wherein the region encoding the α-globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484,229499, 229527,229535 or any combination of two or more of the above base pairs; preferably, the region encoding the α-globin gene contains at least the following positions selected from human chromosome 16 Any one of the base pairs: 229484, 229499 or a combination of the above base pairs.
20.根据16-19任一项所述的试剂盒，其中，所述样本选自全血、外周血、血浆、血清、尿液、唾液、汗液，胎盘、胎盘绒毛、羊水或脐血，或其组合。20. The kit according to any one of 16-19, wherein the sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid or cord blood, or Its combination.
21.根据16-20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。21. The kit according to any one of 16-20, wherein the DNA methylation status display agent is selected from an antibody or a binding protein that recognizes methylated DNA, bisulfite, and has a catalytic oxidation of DNA Enzymes, enzymes with DNA deamination or methylation sensitive enzymes, or a combination thereof; preferably, the methylation sensitive enzyme is selected from methylation sensitive restriction enzymes, more preferably, The methylation-sensitive restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
22.根据16-21任一项所述的试剂盒，其中，所述富集特征游离DNA所需的试剂选自液相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。22. The kit according to any one of 16-21, wherein the reagent required for enriching characteristic free DNA is selected from reagents required for the liquid phase hybridization probe capture method, polymerase chain reaction amplification method The reagents required, the reagents required for the anchored nucleic acid amplification method, the reagents for sequencing-by-synthesis detection, or the reagents for single molecule sequencing, or a combination thereof.
23.一种检测胎儿是否患有遗传疾病或患有遗传疾病的风险度的方法，所述方法包括下列步骤：23. A method for detecting whether a fetus has a genetic disease or a risk of having a genetic disease, the method comprising the following steps:
检测步骤：检测处理后的生物样本中总DNA片段的DNA甲基化模式；其中，所述的生物样本为怀孕女性血浆样本；Detection step: detecting the DNA methylation pattern of the total DNA fragments in the treated biological sample; wherein, the biological sample is a plasma sample of a pregnant female;
区分步骤：区分所述检测步骤中得到的生物样本中总DNA片段的DNA甲基化的不同模式；Distinguishing step: distinguish different patterns of DNA methylation of the total DNA fragments in the biological sample obtained in the detection step;
计算步骤：计算所述生物样本中携带特定DNA甲基化模式的DNA片段占该区域总DNA片段的比例，找到只存在于胎盘组织中而不存在于未孕女性 血浆中的DNA甲基化模式，即胎儿特异的DNA甲基化模式；Calculation step: Calculate the proportion of DNA fragments carrying a specific DNA methylation pattern in the biological sample to the total DNA fragments in the area, and find the DNA methylation pattern that exists only in placental tissue but not in the plasma of infertile women , That is, fetal-specific DNA methylation pattern;
统计步骤：根据找到的胎儿特异的DNA甲基化模式，统计孕妇血浆DNA样本在不同基因区域上胎儿来源的DNA片段占该区域总DNA片段的比例；Statistical steps: According to the fetal specific DNA methylation pattern found, the proportion of fetal DNA fragments in different gene regions of maternal plasma DNA samples in the total DNA fragments in this region is counted;
比较步骤：比较孕妇血浆DNA样本比对到遗传疾病对应的基因区域内的胎儿来源的DNA片段占该区域总DNA片段数的比例与比对到对照基因区域内的胎儿来源的DNA片段占该区域总DNA片段数的比例；Comparison step: comparing the ratio of the DNA fragments of the pregnant women to the DNA fragments of the fetus in the gene region corresponding to the genetic disease to the total number of DNA fragments in the region and the DNA fragments of the fetus from the control gene region to the region The ratio of the total number of DNA fragments;
可选的，所述方法还包括判断步骤：Optionally, the method further includes a judgment step:
如果遗传疾病对应的基因区域内胎儿来源的DNA片段占总DNA片段数的比例与对照基因区域内胎儿来源的DNA片段占总DNA片段数的比例相比，存在不一致，则胎儿患有遗传疾病或具有高的患有遗传疾病的风险；If there is an inconsistency between the proportion of fetal-derived DNA fragments in the total DNA fragments in the gene region corresponding to the genetic disease and the proportion of fetal-derived DNA fragments in the control gene region, the fetus has a genetic disease or Have a high risk of suffering from genetic diseases;
如果遗传疾病对应的基因区域内胎儿来源的DNA片段占总DNA片段数的比例与对照基因区域内胎儿来源的DNA片段占总DNA片段数的比例相比，不存在不一致，则胎儿未患有遗传疾病或不具有患有遗传疾病的风险或具有低的患有遗传疾病的风险。If the proportion of fetal-derived DNA fragments in the total DNA fragments in the gene region corresponding to the genetic disease is not inconsistent compared to the proportion of fetal-derived DNA fragments in the control gene region in the total DNA fragments, the fetus does not have genetic The disease may or may not have a risk of having a genetic disease or have a low risk of having a genetic disease.
24.根据23所述的方法，其中，所述检测步骤前，可包括处理步骤：使用DNA甲基化状态显示剂处理生物样本。24. The method according to 23, wherein before the detecting step, a processing step may be included: processing a biological sample using a DNA methylation status indicator.
25.根据23-24任一项所述的方法，其中，所述计算步骤中，如果某个比对到遗传疾病对应的基因区域、且携带特定DNA甲基化模式的DNA片段只能在携带正常胎儿的孕妇的生物样本中被观察到，而不能从携带患有缺失型遗传疾病的胎儿的孕妇的生物样本中被观察到，则所述特定DNA甲基化模式的DNA片段只能为胎儿来源。25. The method according to any one of 23-24, wherein in the calculation step, if a certain DNA region corresponding to a genetic disease corresponding to a genetic disease and carrying a specific DNA methylation pattern can only be carried in Observed in biological samples of pregnant women with normal fetuses, but not from biological samples of pregnant women with fetuses with deletion genetic diseases, then the DNA fragments of the specific DNA methylation pattern can only be fetuses source.
26.根据23-25任一项所述的方法，其中，所述遗传疾病为α-地中海贫血。26. The method according to any one of 23-25, wherein the genetic disease is α-thalassemia.
27.根据23-26任一项所述的方法，其中，所述遗传疾病对应的基因区域选自编码α-珠蛋白基因的区域；优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体190000-250000碱基对之间的区域；更优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体如下位置中的任意一个碱基对：202146,202148,202161,202170,202178,202310,202425,205180,205229, 205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合。27. The method according to any one of 23-26, wherein the genetic region corresponding to the genetic disease is selected from the region encoding the α-globin gene; preferably, the region encoding the α-globin gene is selected from The region between 190000-250000 base pairs of human chromosome 16; more preferably, the region encoding the α-globin gene is selected from any one of the base pairs of human chromosome 16 in the following positions: 202146, 202148 ,202161,202170,202178,202310,202425,205180,205229, 205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284 ,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116 ,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527 , 229535 or any combination of two or more of the above base pairs.
28.根据23-27任一项所述的方法，其中，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合；优选的，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499或上述碱基对的组合。28. The method according to any one of 23-27, wherein the region encoding the α-globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484,229499,229527 , 229535 or any combination of two or more of the above base pairs; preferably, the region encoding the α-globin gene contains at least the following positions selected from human chromosome 16 Any base pair: 229484, 229499 or a combination of the above base pairs.
29.根据23-28任一项所述的方法，其中，所述样本选自全血、外周血、血浆、血清、尿液、唾液、汗液，胎盘、胎盘绒毛、羊水或脐血，或其组合。29. The method according to any one of 23-28, wherein the sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid or cord blood, or combination.
30.根据23-29任一项所述的方法，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。30. The method according to any one of 23-29, wherein the DNA methylation status display agent is selected from an antibody or binding protein that recognizes methylated DNA, bisulfite, and an enzyme that has catalytic oxidation of DNA , Enzymes with DNA deamination or methylation-sensitive enzymes, or a combination thereof; preferably, the methylation-sensitive enzyme is selected from methylation-sensitive restriction enzymes, more preferably, the The methylation-sensitive restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
31.根据23-30任一项所述的方法，其中，所述富集特征游离DNA所需的试剂选自液相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。31. The method according to any one of 23-30, wherein the reagent required for enriching the characteristic free DNA is selected from the reagents required for the liquid phase hybridization probe capture method and the polymerase chain reaction amplification method. Required reagents, reagents required for the anchoring nucleic acid amplification method, reagents for sequencing-by-synthesis detection, or reagents for single-molecule sequencing, or a combination thereof.
32.一种检测胎儿是否患有遗传疾病或患有遗传疾病的风险度的检测系统，其中，所述检测系统包括如下模块：32. A detection system for detecting whether a fetus has a genetic disease or a risk of having a genetic disease, wherein the detection system includes the following modules:
检测模块：所述检测模块检测处理后的生物样本中总DNA片段的DNA甲基化模式；其中，所述的生物样本为怀孕女性血浆样本；Detection module: the detection module detects the DNA methylation pattern of the total DNA fragments in the processed biological sample; wherein, the biological sample is a plasma sample of a pregnant female;
区分模块：所述区分模块区分所述检测步骤中得到的生物样本中总DNA片段的DNA甲基化的不同模式；Differentiation module: The differentiation module distinguishes different patterns of DNA methylation of the total DNA fragments in the biological sample obtained in the detection step;
计算模块：所述计算模块计算所述生物样本中携带特定DNA甲基化模式的DNA片段占该区域总DNA片段的比例，找到只存在于胎盘组织中而不存在于未孕女性血浆中的DNA甲基化模式，即胎儿特异的DNA甲基化模式；Calculation module: The calculation module calculates the proportion of DNA fragments carrying a specific DNA methylation pattern in the biological sample to the total DNA fragments in the region, and finds DNA that exists only in placental tissue but not in the plasma of infertile women Methylation mode, that is, fetal-specific DNA methylation mode;
统计模块：所述统计模块根据找到的胎儿特异的DNA甲基化模式，统计孕妇血浆DNA样本在不同基因区域上胎儿来源的DNA片段占该区域总DNA片段的比例；Statistic module: The statistic module counts the proportion of fetal-derived DNA fragments of different maternal plasma DNA samples in different gene regions to the total DNA fragments in the region based on the found fetal-specific DNA methylation patterns;
比较模块：所述比较模块比较孕妇血浆DNA样本比对到遗传疾病对应的基因区域内的胎儿来源的DNA片段占该区域总DNA片段数的比例与比对到对照基因区域内的胎儿来源的DNA片段占该区域总DNA片段数的比例；Comparison module: the comparison module compares the ratio of the DNA fragments of the fetus in the gene region corresponding to the genetic disease to the DNA fragments of the fetus in the genetic region corresponding to the genetic disease and the DNA from the fetus in the control gene region The proportion of fragments in the total number of DNA fragments in the region;
可选的，所述系统还可以包括判断模块：Optionally, the system may further include a judgment module:
所述判断模块进行如下判断：The judgment module makes the following judgments:
如果遗传疾病对应的基因区域内胎儿来源的DNA片段占总DNA片段数的比例与对照基因区域内胎儿来源的DNA片段占总DNA片段数的比例相比，存在不一致，则胎儿患有遗传疾病或具有高的患有遗传疾病的风险；If there is an inconsistency between the proportion of fetal-derived DNA fragments in the total DNA fragments in the gene region corresponding to the genetic disease and the proportion of fetal-derived DNA fragments in the control gene region, the fetus has a genetic disease or Have a high risk of suffering from genetic diseases;
如果遗传疾病对应的基因区域内胎儿来源的DNA片段占总DNA片段数的比例与对照基因区域内胎儿来源的DNA片段占总DNA片段数的比例相比，不存在不一致，则胎儿未患有遗传疾病或不具有患有遗传疾病的风险或具有低的患有遗传疾病的风险。If the proportion of fetal-derived DNA fragments in the total DNA fragments in the gene region corresponding to the genetic disease is not inconsistent compared to the proportion of fetal-derived DNA fragments in the control gene region in the total DNA fragments, the fetus does not have genetic The disease may or may not have a risk of having a genetic disease or have a low risk of having a genetic disease.
33.根据32所述的检测系统，其中，所述检测系统还包括处理模块：所述处理模块使用DNA甲基化状态显示剂处理生物样本。33. The detection system according to 32, wherein the detection system further comprises a processing module: the processing module processes a biological sample using a DNA methylation status indicator.
34.根据32-33任一项所述的检测系统，其中，所述计算模块中，如果某个比对到遗传疾病对应的基因区域、且携带特定DNA甲基化模式的DNA片段只能在携带正常胎儿的孕妇的生物样本中被观察到，而不能从携带患有缺 失型遗传疾病的胎儿的孕妇的生物样本中被观察到，则所述特定DNA甲基化模式的DNA片段只能为胎儿来源。34. The detection system according to any one of 32-33, wherein, in the calculation module, if a certain DNA segment corresponding to a genetic region corresponding to a genetic disease and carrying a specific DNA methylation pattern can only be located in Observed in biological samples of pregnant women carrying normal fetuses, but not from biological samples of pregnant women carrying fetuses with missing genetic diseases, the DNA fragments of the specific DNA methylation pattern can only be Fetal source.
35.根据32-34任一项所述的检测系统，其中，所述遗传疾病为α-地中海贫血。35. The detection system according to any one of 32-34, wherein the genetic disease is α-thalassemia.
36.根据32-35任一项所述的检测系统，其中，所述遗传疾病对应的基因区域选自编码α-珠蛋白基因的区域；优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体190000-250000碱基对之间的区域；更优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体如下位置中的任意一个碱基对：202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合。36. The detection system according to any one of 32-35, wherein the genetic region corresponding to the genetic disease is selected from the region encoding the α-globin gene; preferably, the region encoding the α-globin gene is selected From the region between 190000-250000 base pairs of human chromosome 16; more preferably, the region encoding the α-globin gene is selected from any one of the base pairs of human chromosome 16 in the following position: 202146, 202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278, 210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104, 213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499, 229527,229535 or any combination of two or more of the above base pairs.
37.根据32-36任一项所述的检测系统，其中，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合；优选的，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499或上述碱基对的组合。37. The detection system according to any one of 32-36, wherein the region encoding the α-globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484,229499, 229527,229535 or any combination of two or more of the above base pairs; preferably, the region encoding the α-globin gene contains at least the following positions selected from human chromosome 16 Any one of the base pairs: 229484, 229499 or a combination of the above base pairs.
38.根据32-37任一项所述的检测系统，其中，所述样本选自全血、外周血、血浆、血清、尿液、唾液、汗液，胎盘、胎盘绒毛、羊水或脐血，或其组合。38. The detection system according to any one of 32-37, wherein the sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid or cord blood, or Its combination.
39.根据32-38任一项所述的检测系统，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。39. The detection system according to any one of 32-38, wherein the DNA methylation status display agent is selected from an antibody or a binding protein that recognizes methylated DNA, bisulfite, and has a catalytic oxidation of DNA Enzymes, enzymes with DNA deamination or methylation sensitive enzymes, or a combination thereof; preferably, the methylation sensitive enzyme is selected from methylation sensitive restriction enzymes, more preferably, The methylation-sensitive restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
40.根据32-39任一项所述的检测系统，其中，所述富集特征游离DNA所需的试剂选自液相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。40. The detection system according to any one of 32-39, wherein the reagent required for enriching characteristic free DNA is selected from reagents required for liquid phase hybridization probe capture method, polymerase chain reaction amplification method The reagents required, the reagents required for the anchored nucleic acid amplification method, the reagents for sequencing-by-synthesis detection, or the reagents for single molecule sequencing, or a combination thereof.
41.一种用于检测胎儿是否患有遗传疾病或患有遗传疾病的风险度的检测设备，包括：41. A detection device for detecting whether a fetus has a genetic disease or a risk of genetic disease, including:
处理器；processor;
用于存储处理器和执行指令的存储器；Memory for storing processors and executing instructions;
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，实现前述23-31任一项所述的方法。Wherein, the processor is configured to implement the method according to any one of the foregoing 23-31 when executing the processor executable instructions.
42.一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，实现前述23-31任一项所述的方法。42. A non-volatile computer-readable storage medium on which computer program instructions are stored, and when the computer program instructions are executed by a processor, implement the method of any one of the foregoing 23-31.
本公开的一个方面，提供了一种检测胎儿是否患有遗传疾病或患有遗传疾病的风险度的方法，所述方法包括下列步骤：检测处理后的生物样本中总DNA片段的DNA甲基化模式；区分所述检测步骤中得到的生物样本中总DNA片段的DNA甲基化的不同模式；计算所述生物样本中携带特定DNA甲基化模式的DNA片段占该区域总DNA片段的比例，找到只存在于胎盘组织中而不存在于未孕女性血浆中的DNA甲基化模式，即胎儿特异的DNA甲基化模式；根据找到的胎儿特异的DNA甲基化模式，统计孕妇血浆DNA样本在不同基因区域上胎儿来源的DNA片段占该区域总DNA片段的比例；比较孕妇血浆DNA样本比对到遗传疾病对应的基因区域内的胎儿来源的DNA片段占该区域总DNA片段数的比例与比对到对照基因区域内的胎儿来源的DNA片段占该区域总DNA片段数的比例。在优选的实施方案中，通过判断 前述比例的异同，来检测胎儿是否患有遗传疾病或患有遗传疾病的风险度。An aspect of the present disclosure provides a method for detecting whether a fetus has a genetic disease or a risk of suffering from a genetic disease, the method comprising the following steps: detecting DNA methylation of total DNA fragments in a treated biological sample Pattern; distinguish the different patterns of DNA methylation of the total DNA fragments in the biological sample obtained in the detection step; calculate the proportion of DNA fragments carrying a specific DNA methylation pattern in the biological sample to the total DNA fragments in the region, Find the DNA methylation pattern that exists only in placental tissue but not in the plasma of infertile women, that is, fetal-specific DNA methylation pattern; according to the found fetal-specific DNA methylation pattern, count the plasma DNA samples of pregnant women In different gene regions, the proportion of fetal-derived DNA fragments in the total DNA fragments of the region; comparing the ratio of maternal plasma DNA samples to the fetal-derived DNA fragments in the genetic region corresponding to the genetic disease to the total number of DNA fragments in the region The proportion of fetal-derived DNA fragments in the control gene region to the total number of DNA fragments in the region. In a preferred embodiment, whether the fetus has a genetic disease or a risk of having a genetic disease is detected by judging the similarities and differences of the aforementioned ratios.
本公开的另一个方面，提供了一种检测胎儿是否患有α-地中海贫血或患有α-地中海贫血的风险的方法。所述方法包括下列步骤：检测生物样本中DNA的甲基化模式；区分检测步骤中得到的生物样本中总DNA片段的DNA甲基化的不同模式；计算得到α-珠蛋白基因区域内胎盘组织相比于未孕女性血浆特异的DNA的甲基化模式；统计孕妇血浆DNA在α-珠蛋白基因区域以及对照区域内具有胎儿特异甲基化模式的DNA片段数占该区域内总DNA片段数的比例；比较α-珠蛋白基因区域以及对照区域内具有胎儿特异甲基化模式的DNA片段数占该区域内总DNA片段数的比例。在优选的实施方案中，通过判断前述比例的异同，来检测胎儿是否患有α-地中海贫血。In another aspect of the present disclosure, there is provided a method of detecting whether a fetus has or is at risk of suffering from α-thalassemia. The method includes the following steps: detecting the methylation pattern of DNA in a biological sample; distinguishing different patterns of DNA methylation of the total DNA fragments in the biological sample obtained in the detection step; calculating the placental tissue within the α-globin gene region Compared with the methylation pattern of DNA specific to non-pregnant women's plasma; the number of DNA fragments with fetal-specific methylation pattern in the α-globin gene region and the control region of pregnant women's plasma DNA accounted for the total number of DNA fragments in this region The ratio of α-globin gene region and the control region of the DNA fragments with fetal-specific methylation pattern to the total number of DNA fragments in this region. In a preferred embodiment, whether the fetus has α-thalassemia is determined by judging the similarities and differences of the aforementioned ratios.
在一些实施方案中，检测样本可以是孕妇的外周血、尿液、唾液、汗液。在一些实施方案中，检测样本为孕妇的外周血。进一步地，对孕妇外周血样本进行处理，获得血浆或血清。在一些实施方案中，对血浆样本进行检测。对孕妇的外周血、尿液、唾液、汗液等样本进行检测之后，对胎儿α-珠蛋白基因的基因型进行评估属于无创检测手段。检测样本可以是胎儿组织，例如胎盘组织，检测所述样本则是一种有创检测方案。In some embodiments, the test sample may be a pregnant woman's peripheral blood, urine, saliva, sweat. In some embodiments, the test sample is the peripheral blood of the pregnant woman. Further, the peripheral blood samples of pregnant women are processed to obtain plasma or serum. In some embodiments, the plasma sample is tested. After testing the samples of pregnant women's peripheral blood, urine, saliva, sweat, etc., assessing the genotype of the fetal α-globin gene is a non-invasive detection method. The test sample may be fetal tissue, such as placental tissue, and detecting the sample is an invasive detection scheme.
在一些实施方案中，不需要使用DNA甲基化状态显示剂处理样本。In some embodiments, it is not necessary to treat the sample with a DNA methylation status indicator.
在另一些实施方案中，在获取检测样本后，使用DNA甲基化状态显示剂处理样本。在一些实施方案中，利用DNA甲基化状态显示剂对样本进行处理，甲基化和非甲基化修饰的DNA会显示出差异。DNA甲基化状态显示剂可以是亚硫酸氢盐，甲基化敏感的限制性内切酶(例如HpaII和BstUI)，识别甲基化DNA的抗体或结合蛋白等。在一些实施方案中，利用亚硫酸氢盐对样本进行处理。In other embodiments, after obtaining the test sample, the sample is treated with a DNA methylation status indicator. In some embodiments, the DNA methylation status indicator is used to process the sample, and methylated and non-methylated modified DNA will show differences. The DNA methylation status indicator can be bisulfite, methylation-sensitive restriction enzymes (such as HpaII and BstUI), antibodies or binding proteins that recognize methylated DNA. In some embodiments, the sample is processed using bisulfite.
在一些实施方案中，所述检测样本中DNA的甲基化模式的步骤中使用的DNA甲基化模式的检测方法为测序法。在一些实施方案中，将样本DNA构建为全基因组亚硫酸氢盐测序文库，随后利用基于边合成边测序原理的检测平台，例如下一代测序仪，对所述文库进行测序。可选择地，用于步骤(2) 的检测方法还包括实时PCR、数字PCR、质谱法、单分子测序法等。In some embodiments, the detection method of the DNA methylation pattern used in the step of detecting the methylation pattern of DNA in the sample is a sequencing method. In some embodiments, the sample DNA is constructed as a whole genome bisulfite sequencing library, and then the library is sequenced using a detection platform based on the sequencing-by-synthesis principle, such as a next-generation sequencer. Optionally, the detection method used in step (2) also includes real-time PCR, digital PCR, mass spectrometry, single molecule sequencing, and the like.
在一些实施方案中，在获得样本甲基化模式信息后，统计具有胎儿特异甲基化模式的DNA数目。在一些实施方案中，对检测样本中DNA的甲基化模式的步骤中获得的样本DNA的甲基化模式进行统计，使用甲基化模式来分类DNA片段。正如领域内的工申请人熟知的，该统计分析可以利用连锁分析、一一对应分析等方式实现。在另一些实施方案中，可利用机器学习方法区分检测样本中游离DNA片段上的甲基化模式。In some embodiments, after obtaining sample methylation pattern information, the number of DNAs with fetal-specific methylation patterns is counted. In some embodiments, the methylation pattern of the sample DNA obtained in the step of detecting the methylation pattern of DNA in the sample is counted, and the methylation pattern is used to classify the DNA fragments. As well-known to applicants in the field, this statistical analysis can be implemented using chain analysis, one-to-one correspondence analysis, and so on. In other embodiments, machine learning methods can be used to distinguish methylation patterns on free DNA fragments in the detection sample.
在一些实施方案中，分别计算每条携带特定甲基化模式的DNA片段来自胎盘或来自母体的概率。在一些实施方案中，可以使用数学方式去定义一条DNA片段为胎儿来源的DNA片段，当其来自胎儿的概率，显著地高于来自母体的概率。正如领域内的工申请人熟知的，该统计分析可使用卡方检验、秩和检验、麦当劳检验、超几何检验、普通t检验等统计领域众所周知的技术实现。在另一些实施方案中，可以使用生物学样本作对照比较，例如，某个比对到α-珠蛋白基因区域基因组、且携带特定甲基化模式的DNA片段，只能从一个携带正常胎儿的孕妇外周血游离DNA样本中观察到，且不能从一个携带纯合α-珠蛋白基因区域缺失突变胎儿的孕妇外周血游离DNA样本中被观察到，则认为该甲基化模式只能是来自胎儿基因组的α-珠蛋白基因区域。正如领域内的工申请人熟知的，这类生物学样本可以有多种选取方式，如，选取的生物学样本是孕妇外周血、胎盘样本、胎盘绒毛样本、羊水穿刺样本、脐血样本等，都是可能的。选取不同的生物学样本，并不改变结果。在本步骤完成之后，每条DNA片段可以被唯一地定义为“胎儿来源的DNA片段”或“不一定是胎儿来源的DNA片段”。In some embodiments, the probability that each DNA fragment carrying a specific methylation pattern comes from the placenta or from the mother is calculated separately. In some embodiments, a DNA fragment can be defined mathematically as a DNA fragment of fetal origin, when the probability of it from the fetus is significantly higher than that from the mother. As the applicants in the field are familiar with, this statistical analysis can be implemented using techniques well known in the statistical field such as chi-square test, rank sum test, McDonald's test, hypergeometric test, and general t test. In other embodiments, a biological sample can be used as a control for comparison, for example, a DNA fragment that is aligned to the genome of the α-globin gene region and carries a specific methylation pattern can only be obtained from a It was observed in free DNA samples from pregnant women's peripheral blood and could not be observed from a free blood sample from pregnant women carrying homozygous α-globin gene region deletion mutant fetuses, so the methylation pattern can only be derived from the fetus The α-globin gene region of the genome. As the applicants in the field are familiar with, such biological samples can be selected in multiple ways. For example, the selected biological samples are maternal peripheral blood, placenta samples, placental villi samples, amniocentesis samples, cord blood samples, etc. Are possible. Selecting different biological samples does not change the results. After the completion of this step, each DNA fragment can be uniquely defined as "a DNA fragment of fetal origin" or "not necessarily a DNA fragment of fetal origin".
在一些实施方案中，分析统计具有胎儿特异甲基化模式的DNA数目中，比对到α-珠蛋白基因区域内以及对照区域(下统称“基因组区域”)的DNA。具体地，分析出胎儿来源DNA片段占总DNA片段数的比例。按照统计原理，该比例在不同的基因组区域之间应保持一致。在某基因组区域内，该比例偏离了平均值，则可能代表胎儿基因组的该区域出现了拷贝数变化。进而，通 过比较比对到α-珠蛋白基因区域内的胎儿来源DNA片段占总DNA片段数的比例与比对到对照基因区域内的胎儿来源DNA片段占总DNA片段数的比例，人们便可获知胎儿的α-珠蛋白基因区域是否可能存在拷贝数缺失或重复。In some embodiments, among the number of DNAs with fetal-specific methylation patterns, the DNA in the α-globin gene region and the control region (hereinafter collectively referred to as “genomic regions”) is compared. Specifically, the ratio of fetal DNA fragments to the total number of DNA fragments is analyzed. According to statistical principles, this ratio should be consistent between different genomic regions. In a genomic region, the ratio deviates from the average value, which may represent a copy number change in the region of the fetal genome. Furthermore, by comparing the proportion of fetal DNA fragments in the α-globin gene region to the total number of DNA fragments and the proportion of fetal DNA fragments in the control gene region to the total number of DNA fragments, one can then It is known whether there may be copy number deletion or duplication in the fetal α-globin gene region.
在一些实施方案中，正如领域内工申请人所熟知的，胎儿携带的基因突变和遗传缺陷，可能造成其它基因组区域(下称“关联区域”)的甲基化模式改变。通过比较关联区域的甲基化模式改变，也可推测胎儿携带的基因突变情况。在一些实施方案中，可以以关联区域的甲基化模式改变，推测胎儿是否携带α-珠蛋白基因缺陷。In some embodiments, as well known to applicants in the art, genetic mutations and genetic defects carried by the fetus may cause changes in the methylation pattern of other genomic regions (hereinafter "associated regions"). By comparing changes in methylation patterns in related regions, it is also possible to speculate on genetic mutations carried by the fetus. In some embodiments, the methylation pattern of the associated region can be altered to speculate whether the fetus carries the α-globin gene defect.
所述方法适合于检测胎儿α-地中海贫血病诊断，所述α-地中海贫血病包括α-珠蛋白基因缺失型和突变型。在一些实施方案中，利用所述方法对α-珠蛋白基因全部缺失的胎儿进行诊断。例如，所述胎儿α-珠蛋白基因为SEA缺失型等位基因形成的纯合子(SEA/SEA)。α-珠蛋白基因全部缺失的胎儿表现为贫血伴全身水肿，容易出现宫内死亡，同时会引发孕妇严重的并发症。在一些实施方案中，所述胎儿α-珠蛋白基因为SEA，THAL，α3.7、α4.2缺失型等位基因组合形成的基因型，例如SEA/SEA、SEA/α3.7、SEA/α4.2等。在一些实施例中，所述胎儿α-珠蛋白基因携带点突变，例如αCS、αWS。此外，所述方法还可以对妊娠相关病症的存在或发病风险进行评估。例如，所述妊娠相关病症为早产等。The method is suitable for detecting the diagnosis of fetal α-thalassemia, which includes α-globin gene deletion type and mutant type. In some embodiments, the method is used to diagnose a fetus with a complete deletion of the α-globin gene. For example, the fetal α-globin gene is a homozygote (SEA/SEA) formed by SEA deletion alleles. Fetuses with all alpha-globin genes deleted show anemia with systemic edema, which is prone to intrauterine death and can cause serious complications in pregnant women. In some embodiments, the fetal α-globin gene is a genotype formed by a combination of SEA, THAL, α3.7, and α4.2 deletion alleles, such as SEA/SEA, SEA/α3.7, SEA/ α4.2 etc. In some embodiments, the fetal α-globin gene carries point mutations, such as αCS, αWS. In addition, the method can also assess the presence or risk of pregnancy-related disorders. For example, the pregnancy-related disorder is premature delivery.
本公开的另一个方面，提供了一种检测胎儿是否患有α-地中海贫血的试剂盒。该试剂盒包含富集特征游离DNA所需的试剂。在一些实施方案中，所述试剂盒还可包含DNA甲基化状态显示剂。在一些实施方案中，通过液相杂交探针捕获法富集特征游离DNA。在一些实施方案中，通过聚合酶链式反应扩增法(PCR)富集特征来源游离DNA。在一些实施方案中，通过锚定核酸扩增法(anchor PCR)富集特征来源游离DNA。在一些实施方案中，该试剂盒包括将富集的特征游离DNA用于边合成边测序检测的试剂(即所谓的建库试剂)。在一些实施方案中，该试剂盒包括将游离DNA用于单分子测序检测 的试剂(即所谓的建库试剂)。在一些实施方案中，还提供对特征游离DNA进行统计分析的软件，将α-珠蛋白基因区域内胎儿来源DNA分数与标准对照进行比较，判断胎儿α-珠蛋白基因位点的基因型。In another aspect of the present disclosure, a kit for detecting whether a fetus has α-thalassemia is provided. The kit contains the reagents needed to enrich the characteristic free DNA. In some embodiments, the kit may further comprise a DNA methylation status indicator. In some embodiments, characteristic free DNA is enriched by liquid phase hybridization probe capture. In some embodiments, characteristic source free DNA is enriched by polymerase chain reaction amplification (PCR). In some embodiments, the characteristic source free DNA is enriched by anchor PCR. In some embodiments, the kit includes reagents that use the enriched characteristic free DNA for sequencing-by-synthesis detection (so-called library building reagents). In some embodiments, the kit includes reagents that use free DNA for single molecule sequencing detection (i.e., so-called library-building reagents). In some embodiments, software for statistical analysis of characteristic free DNA is also provided to compare the fraction of fetal DNA in the α-globin gene region with a standard control to determine the genotype of the fetal α-globin gene locus.
发明的效果Effect of invention
在本公开的一个实施方案中，本公开发现，在珠蛋白基因所在基因组区域(例如，人类16号染色体190000-250000碱基对之间的区域)，胎儿DNA具有特异的甲基化模式，所述甲基化模式与母体DNA的甲基化模式不同。In one embodiment of the present disclosure, the present disclosure finds that fetal DNA has a specific methylation pattern in the genomic region where the globin gene is located (for example, the region between 190000-250000 base pairs of human chromosome 16). The methylation pattern is different from that of the maternal DNA.
在本公开的另一个实施方案中，本公开提供了许多全新的，胎儿高度特异的DNA甲基化标记物。所述标记物可以在单分子水平上区分母源DNA和胎源DNA，进而提供对胎儿来源的DNA片段进行独立分析的方法。因此，本公开还进一步提供了一种更加准确、更加高效、更加简便的非侵入性诊断胎儿α-地中海贫血的方法。所述方法在本公开提供的试验中表现出高度准确性。本公开还提供了胎儿α-地中海贫血产前诊断的试剂盒。In another embodiment of the present disclosure, the present disclosure provides many new, highly specific DNA methylation markers for the fetus. The marker can distinguish maternal DNA and fetal DNA at a single molecule level, thereby providing a method for independent analysis of fetal-derived DNA fragments. Therefore, the present disclosure further provides a more accurate, more efficient, and more convenient method for non-invasive diagnosis of fetal α-thalassemia. The method has shown a high degree of accuracy in the experiments provided by the present disclosure. The present disclosure also provides a kit for prenatal diagnosis of fetal α-thalassemia.
在本公开的另一个实施方案中，本公开提供的胎儿特异的甲基化标记物还可用于判断母体游离DNA中胎儿DNA的存在以及含量，并进一步用于胎儿其他病症以及孕妇妊娠相关病症或患病风险的评估。In another embodiment of the present disclosure, the fetal-specific methylation markers provided by the present disclosure can also be used to determine the presence and content of fetal DNA in maternal free DNA, and further used for other fetal disorders and pregnancy-related disorders of pregnant women or Assessment of disease risk.
附图说明BRIEF DESCRIPTION
图1A-图1C所示的是通过全基因组甲基化测序，发现的胎儿与母体差异甲基化位点。其中，图1A示出了每个胎盘样本与母体样本之间均存在稳定的甲基化(黑：高；白：低)水平差异。图1B示出了前述的胎儿与母体差异甲基化位点，在每条染色体上均存在。图1C示出了前述的胎儿与母体差异甲基化位点中，部分为同一基因组区域内分布的连续多个CpG位点。Figures 1A-1C show the differential methylation sites between the fetus and the mother found through whole-genome methylation sequencing. Among them, FIG. 1A shows that there is a stable level of methylation (black: high; white: low) between each placenta sample and the maternal sample. FIG. 1B shows the aforementioned differential methylation sites of fetus and mother, which are present on each chromosome. FIG. 1C shows the continuous multiple CpG sites distributed in the same genomic region among the aforementioned fetal and maternal differential methylation sites.
图2所示的是通过全基因组甲基化测序，发现的胎儿与母体差异单分子甲基化标志物。其中，图2中的A部分示出了在该示例基因组位置上，胎盘样本与母体样本之间，在每个CpG位点上均存在稳定的甲基化(黑：高；白：低)水平差异，同时可观察到胎盘特异的单分子甲基化标志物(具白色色块 的分子)及母体特异的单分子甲基化标志物(具黑色色块的分子)。图2中的B部分示出了在该示例基因组位置上，胎盘样本与母体样本之间，不一定在每个CpG位点上均存在稳定的甲基化(黑：高；白：低)水平差异，但仍旧观察到胎盘特异的单分子甲基化标志物(具白色色块的分子)Figure 2 shows the single-molecule methylation markers of fetal and maternal differences discovered through whole-genome methylation sequencing. Among them, part A in Figure 2 shows that at this exemplary genomic location, there is a stable level of methylation (black: high; white: low) at each CpG site between the placenta sample and the maternal sample Differences, at the same time, the placenta-specific single-molecule methylation marker (molecule with white color block) and the maternal-specific single-molecule methylation marker (molecule with black color block) can be observed. Part B in Figure 2 shows that at this exemplary genomic location, there is not necessarily a stable level of methylation (black: high; white: low) at each CpG site between the placenta sample and the maternal sample Differences, but single-molecule methylation markers specific to the placenta (molecules with white patches) are still observed
图3所示的是通过全基因组甲基化测序，发现α-珠蛋白基因组区域的胎儿特异的甲基化标志物。其中，图3中的A部分示出了胎盘样本与母体样本之间,存在特异的单分子甲基化标志物(黑：高；白：低)差异。整条DNA分子上全为去甲基化模式的DNA，只能在胎盘样本中发现。图3中的B部分示出了前述特异的单分子甲基化标志物，只在α-珠蛋白基因组区域为野生型或杂合携带SEA缺失基因型的绒毛中可以被观察到，在纯合SEA缺失基因型的绒毛和未孕女性外周血中，并不能被观察到。该实验证明了，该单分子甲基化标志物，只能来自于胎儿基因组，不可能来自于母亲基因组，是特异的α-珠蛋白基因组区域胎儿来源DNA片段标记物。特别地，该家族的单分子甲基化标志物，位于人类16号染色体190000-230000碱基对(GRCh37版本基因组)区域内。更特别地，该家族的单分子甲基化标志物，包含表6中的CpG位点及甲基化状态组合。Figure 3 shows fetal-specific methylation markers found in the α-globin genomic region through whole-genome methylation sequencing. Among them, Part A in FIG. 3 shows that there are specific single-molecule methylation markers (black: high; white: low) differences between the placenta sample and the maternal sample. The entire DNA molecule is all demethylated DNA and can only be found in placental samples. Part B in Figure 3 shows the aforementioned specific single-molecule methylation markers, which can only be observed in the villi where the α-globin genomic region is wild-type or heterozygous carrying the SEA-deleted genotype. SEA-deficient genotype villi and infertile women's peripheral blood cannot be observed. This experiment proves that the single-molecule methylation marker can only come from the fetal genome, but not from the maternal genome. It is a specific DNA fragment marker of fetal origin in the α-globin genome region. In particular, this family of single-molecule methylation markers is located in the region of 190000-230000 base pairs (GRCh37 version genome) of human chromosome 16. More specifically, the single-molecule methylation markers of this family include CpG sites and methylation status combinations in Table 6.
图4所示的是在68例孕妇血浆中，携带纯合SEA缺失基因型胎儿的孕妇，其归一化后的α-珠蛋白基因组区域局部胎源比例显著地低于其他孕妇。按照0.01的归一化后局部胎源比例划线，灵敏度为100％，特异性为96％。Figure 4 shows that in the plasma of 68 pregnant women carrying pregnant women with homozygous SEA-deficient genotypes, their normalized α-globin genomic region has a significantly lower local fetal proportion than other pregnant women. According to the normalized proportion of local fetus after 0.01, the sensitivity is 100% and the specificity is 96%.
图5所示的是利用胎盘特异的甲基化模式，测试胎儿基因型未知的孕妇血浆DNA的检测结果。Figure 5 shows the results of testing the plasma DNA of pregnant women with unknown fetal genotype using the specific methylation pattern of the placenta.
具体实施方案Specific implementation plan
定义definition
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”。When used in conjunction with the term "comprising" in the claims and/or description, the words "a" or "an" may mean "a", but may also mean "one or more", "at least "One" and "one or more than one".
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。与此同时，“包含”、“具有”、“包括”或“含有”也可以表示封闭式的，排除额外的、未引述的元件或方法步骤。As used in the claims and the description, the words "include", "have", "include" or "include" mean inclusive or open-ended and do not exclude additional, unquoted elements or methods step. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, unquoted elements or method steps.
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。Throughout the application document, the term "about" means that a value includes the standard deviation of the error of the device or method used to determine the value.
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，权利要求中的术语“或”是指“和/或”。Although the disclosed content supports the definition of the term "or" as only a substitute and "and/or", unless explicitly stated that the substitute or the substitutes are mutually exclusive, the term "or" in the claims means "and / or".
“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。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甲基化”通常是指核苷酸胞嘧啶残基的5’碳的甲基化，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-羟基甲基胞嘧啶等形式。"DNA methylation" usually refers to the methylation of the 5'carbon of the nucleotide cytosine residue. 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 a phosphate group. DNA methylation can also occur in cytosines of nucleotide sequences such as CHG and CHH, where H is adenine, cytosine or thymine. DNA methylation can also occur on non-cytosine, such as N6-methyladenine. In addition, DNA methylation can also be in the form of 5-hydroxymethylcytosine.
“甲基化位点”是指可能发生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，甲基化位点等同于CpG位点。"Methylation site" refers to a single or multiple base positions where methylation modification may occur. For example, CpG site, CHG site or CHH site. In some cases, the methylation site is equivalent to the CpG site.
“甲基化模式”是对样本DNA在所述区域内整体甲基化状态的描述。可以是“甲基化状态”、“甲基化水平”、“甲基化密度”或“单分子甲基化状态”以及它们的组合。"Methylation mode" is a description of the overall methylation status of the sample DNA in the region. It can be "methylation state", "methylation level", "methylation density" or "single molecule methylation state" and combinations thereof.
“甲基化状态”是指DNA区段的特定基因组基因座与甲基化相关的特征。此类特征包括但不限于：该DNA序列内是否有任何胞嘧啶(C)残基被甲基化，甲基化C残基的位置，残基任何特定延伸段处甲基化C的百分比，和由于(例如)等位基因来源的差异导致的等位基因差异。术语“甲基化模式” 或“甲基化状况”也指生物学样品中任何特定残基延伸段的甲基化C或未甲基化C的相对或绝对浓度。例如，如果DNA序列内有一个或多个胞嘧啶(C)残基甲基化，可称其为“高甲基化”；如果DNA序列内一个或多个胞嘧啶(C)残基未甲基化，可称其为“低甲基化”。类似地，如果DNA序列(例如，胎儿核酸)内一个或多个胞嘧啶(C)残基相对于不同来源或不同个体的另一序列(例如，相对于母体核酸)被甲基化，则该序列相对于该另一序列视作高甲基化。或者，如果DNA序列内的一个或多个胞嘧啶(C)残基相对于不同来源或不同个体(例如，母亲)的另一序列未被甲基化，则该序列相对于该另一序列视作低甲基化。将这些序列称为“差异甲基化”，更具体地说，当母亲与胎儿之间的甲基化状态不同时，这些序列视作“差异甲基化的母体和胎儿核酸”。"Methylation status" refers to a feature of a particular genomic locus of a DNA segment that is associated with methylation. Such characteristics include, but are not limited to, whether any cytosine (C) residues in the DNA sequence are methylated, the position of the methylated C residue, and the percentage of methylated C at any particular extension of the residue, And allelic differences due to, for example, differences in allelic origin. The term "methylation mode" or "methylation status" also refers to the relative or absolute concentration of methylated C or unmethylated C of any particular residue extension in a biological sample. For example, if one or more cytosine (C) residues in the DNA sequence are methylated, it can be called "hypermethylation"; if one or more cytosine (C) residues in the DNA sequence are not methylated , Can be called "low methylation". Similarly, if one or more cytosine (C) residues within a DNA sequence (eg, fetal nucleic acid) are methylated relative to another sequence (eg, relative to maternal nucleic acid) from a different source or individual, then the The sequence is considered hypermethylated relative to the other sequence. Alternatively, if one or more cytosine (C) residues within the DNA sequence are not methylated with respect to another sequence from a different source or different individual (eg, mother), then the sequence is viewed relative to the other sequence For low methylation. These sequences are called "differential methylation", and more specifically, when the methylation status between the mother and the fetus is different, these sequences are regarded as "differentially methylated maternal and fetal nucleic acids".
“甲基化水平”是指该甲基化位点上被甲基化修饰的DNA分子的数目除以含有所述位点的总DNA分子数目的数值，用以描述DNA样本中某个甲基化位点的整体甲基化状态。"Methylation level" refers to the number of methylated DNA molecules at the methylation site divided by the number of total DNA molecules containing the site to describe a methyl group in the DNA sample The overall methylation status of the chemical site.
“甲基化密度”是区域内展示甲基化的位点的数目除以所述区域中覆盖位点的读数的总数。"Methylation density" is the number of sites in a region that exhibit methylation divided by the total number of readings covering the sites in the region.
“单分子甲基化状态”是指单个DNA分子具有的所有可被甲基化的碱基位点的甲基化状态组合。单分子甲基化状态可包括1个、两个或多个甲基化位点。例如，具有两个甲基化位点的DNA分子则可能具有4种单分子甲基化状态，包括+-、-+、--和++(“+”表示甲基化位点被甲基化修饰，“-”表示甲基化位点未被甲基化修饰)。在本公开中，单分子甲基化状态用以区分怀孕女性血浆样本中的母源和胎源DNA。“单分子甲基化状态”可以包括至少1个、至少两个、至少3个、至少4个、至少5个、至少6个、至少10个、至少20个、至少40个、至少50个、至少100个、至少300个、至少400个、至少500个或大于500个、或者上述值的任两者之间范围的甲基化位点的甲基化状态。"Single-molecule methylation state" refers to a combination of methylation states of all base sites that can be methylated by a single DNA molecule. The single molecule methylation state may include one, two or more methylation sites. For example, a DNA molecule with two methylation sites may have four single-molecule methylation states, including +-, -+, --, and ++ ("+" indicates that the methylation site is methylated Modified, "-" indicates that the methylation site is not modified by methylation). In the present disclosure, single molecule methylation status is used to distinguish maternal and fetal DNA in plasma samples of pregnant women. "Single molecule methylation state" may include at least 1, at least two, at least 3, at least 4, at least 5, at least 6, at least 10, at least 20, at least 40, at least 50, The methylation status of at least 100, at least 300, at least 400, at least 500 or more, or any range of the methylation sites of the above values.
“DNA甲基化状态显示剂”是指能够差异修饰所述样品中的甲基化和非甲基化DNA的试剂。"DNA methylation status indicator" refers to an agent capable of differentially modifying methylated and unmethylated DNA in the sample.
“检测标记物”是指可以标记系统、器官、组织、细胞及亚细胞结构或功能的改变或可能发生的改变的生化指标。在本公开的一个实施方案中，所述检测标记物为DNA单分子甲基化状态。"Detection marker" refers to a biochemical indicator that can mark changes or possible changes in the structure or function of systems, organs, tissues, cells and sub-cells. In one embodiment of the present disclosure, the detection marker is a single-molecule methylation state of DNA.
“样本”是指来自于人体并含有DNA分子的任何样本。用于胎儿检测的“样本”可以是外周血样本、胎盘样本、胎盘绒毛样本、羊水穿刺样本、脐血样本、尿液样本、唾液样本、汗液样本等。"Sample" refers to any sample that comes from the human body and contains DNA molecules. The "sample" used for fetal detection may be a peripheral blood sample, a placenta sample, a placental villi sample, an amniotic fluid puncture sample, a cord blood sample, a urine sample, a saliva sample, a sweat sample, and the like.
“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。"Fetus" refers to the embryo after 8 weeks of gestational age. "Gestational age" is a measure of gestational age, where the starting point is the corresponding age estimated by the woman's last normal menstrual period (LMP) or other methods. In human obstetrics, gestational age refers to the age of the embryo or fetus plus two weeks. In humans, although delivery is common within 37 to 42 weeks, delivery usually occurs around 40 weeks of gestational age.
“局部胎源比例”是指胎儿来源DNA片段占检测区域上所有DNA片段的比例。"Local fetal proportion" refers to the proportion of fetal DNA fragments to all DNA fragments on the detection area.
“胎儿特异甲基化模式”是指只存在于胎盘组织中而不存在于未孕女性血浆中的DNA甲基化模式。"Fetal-specific methylation pattern" refers to the DNA methylation pattern that exists only in placental tissue but not in the plasma of infertile women.
“α-地中海贫血”是指由α-珠蛋白基因缺陷导致的一种遗传性溶血性贫血。α-地中海贫血的基因缺陷主要由α-珠蛋白基因缺失导致。α-珠蛋白基因簇位于16号染色体上，包括α1和α2两个高度同源的α-珠蛋白基因，α1和α2基因均能表达α-珠蛋白链。一条染色体上的α-珠蛋白基因正常，则记为(αα/)，那么，正常人的基因型则为(αα/αα)。一条染色体上缺失一个α-基因记为(-α/)，例如(-α 3.7/)、(-α 4.2/)等。一条染色体缺失两个α-基因则不能合成α-珠蛋白链，称为α 0-地中海贫血，记为(--/)，例如(-- SEA/)、(-- THAI/)和(-- FIL/)等，其纯合子表现为重型α-地中海贫血，受累胎儿易发生宫内死亡。非缺失型α-地中海贫血一般不会导致一条染色体上的两个α-基因同时受损，因此称为α +-地中海贫血，其等位基因记为(α Mα/)或(αα M/)，例如在中国常见的(α WSα/)、(α CSα/)、(α QSα/)。 "Α-thalassemia" refers to an inherited hemolytic anemia caused by defects in the α-globin gene. The genetic defect of α-thalassemia is mainly caused by the deletion of α-globin gene. The α-globin gene cluster is located on chromosome 16 and includes two highly homologous α-globin genes, α1 and α2, both of which can express α-globin chains. If the α-globin gene on a chromosome is normal, it is recorded as (αα/), then the genotype of a normal person is (αα/αα). The deletion of an α-gene on a chromosome is recorded as (-α/), for example (-α 3.7 /), (-α 4.2 /), etc. If two α-genes are missing from one chromosome, the α-globin chain cannot be synthesized, which is called α 0 -thalassemia, and is denoted as (--/), such as (-- SEA /), (-- THAI /) and (- -FIL /), etc., the homozygote shows severe α-thalassemia, and the affected fetus is prone to intrauterine death. Non-deleted α-thalassemia generally does not cause two α-genes on a chromosome to be damaged at the same time, so it is called α + -thalassemia, and its allele is recorded as (α M α/) or (αα M / ), such as (α WS α/), (α CS α/), (α QS α/) common in China.
“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。"Whole-genome bisulfite sequencing method" is also called WGBS (Whole-genome bisulfite sequencing). Its principle is to process the sample to be tested with bisulfite and convert the unmethylated C bases into U in the genome. After PCR amplification, it becomes T, which is distinguished from the original C base with methylation modification, combined with high-throughput sequencing technology, and compared with the reference sequence, you can judge whether the CpG/CHG/CHH site has a Basalization, especially suitable for mapping whole-genomic DNA methylation maps with single-base resolution.
DNA甲基化状态的检测Detection of DNA methylation status
诸多方法都可以用于DNA的甲基化状态的检测，例如甲基化DNA免疫沉淀测序法(MeDIP-Seq，Jacinto et al.,2008 44(1),35–43)、简化代表性亚硫酸氢盐测序法(RRBS，Meissner et al.,Nucleic Acids Res,2005；33:5868–5877.)、甲基化微阵列(例如，Infinium Human Methylation 450K BeadChip，Sandoval et al.,Epigenetics,2011,6(6),692–702)和全基因组亚硫酸氢盐测序法(WGBS，Lister et al.,Cell,2008；133:523–36.)等。Many methods can be used to detect the methylation status of DNA, such as methylated DNA immunoprecipitation sequencing (MeDIP-Seq, Jacinto et al., 2008 44(1), 35–43), to simplify representative sulfurous acid Hydrogen salt sequencing (RRBS, Meissner et al., Nucleic Acids Res, 2005; 33:5868–5877.), methylated microarrays (for example, Infinium Human Methylation 450K BeadChip, Sandoval et al., Epigenetics, 2011, 6 (6), 692–702) and genome-wide bisulfite sequencing (WGBS, Lister et al., Cell, 2008; 133:523–36.), etc.
在一个实施方案中，全基因组亚硫酸氢盐测序法能够分析基因组上的所有甲基化位点，并且可以在单碱基分辨率条件下对单个DNA分子进行检测。因此，所述方法可以在全基因组范围内全面地研究DNA甲基化状态、并提供DNA甲基化状态的定量分析方法。进一步，所述方法可用于单分子甲基化状态评估。In one embodiment, the whole genome bisulfite sequencing method can analyze all methylation sites on the genome, and can detect a single DNA molecule at a single base resolution condition. Therefore, the method can comprehensively study the DNA methylation status within the whole genome and provide a quantitative analysis method for the DNA methylation status. Further, the method can be used for single molecule methylation status assessment.
近年来，全基因组亚硫酸氢盐测序法已经被成功应用于人类表观遗传学的研究(Lister et al.,Nature,2009,462(7271):315–322；Laurent et al.,Genome Research,2010,20(3):320-331；Kundaje et al.,Nature,2015,518(7539):317–330.)，例如，对胎盘组织的甲基化模式的研究。(Lun et al.,ClinChem,2013,59(11):1583–1594；Jensen et al.,Genome Biol,2015,16(1):78.)。正如上文提到的，利用胎儿特异的DNA甲基化模式就能够达到区别母源和胎源DNA的目的。因此，可以选择采用全基因组亚硫酸氢盐测序法实现本公开所述的方法。In recent years, whole-genome bisulfite sequencing has been successfully applied to the study of human epigenetics (Lister et al., Nature, 2009, 462 (7271): 315–322; Laurent et al., Genome Research, 2010,20(3):320-331; Kundaje et al., Nature,2015,518(7539):317-330.), for example, the study of methylation patterns of placental tissue. (Lun et al., ClinChem, 2013, 59(11): 1583–1594; Jensen et al., Genome Biol, 2015, 16(1): 78.). As mentioned above, the use of fetal-specific DNA methylation patterns can achieve the purpose of distinguishing maternal and fetal DNA. Therefore, the whole genome bisulfite sequencing method can be selected to implement the method described in the present disclosure.
α-珠蛋白基因相关的胎儿DNA标记物鉴定Identification of fetal DNA markers related to α-globin gene
在一个实施方案中，利用全基因组亚硫酸氢盐测序法对胎盘组织DNA和非孕女性血浆游离DNA的甲基化模式进行检测，通过分析所述DNA的单 分子甲基化状态，筛选出在包括但不限于α-珠蛋白基因区域中，所述胎盘组织DNA具有的特异单分子甲基化状态。In one embodiment, the whole-genome bisulfite sequencing method is used to detect the methylation pattern of placental tissue DNA and non-pregnant female plasma free DNA. By analyzing the single-molecule methylation status of the DNA, the Including but not limited to the α-globin gene region, the placental tissue DNA has a specific single molecule methylation state.
在一个实施方案中，如图1所示，在所述α-珠蛋白所在基因组区域上，单个碱基位点的甲基化水平具有极大的噪音，不适用于单分子检测。然而，通过单分子甲基化模式分析，可发现胎盘组织来源的DNA，与非孕女性血浆游离DNA的单分子甲基化模式存在极大差异。进一步地，在另一个实施方案中，使用全基因组亚硫酸氢盐测序法对基因型为αα/αα、-- SEA/αα和-- SEA/-- SEA的胎盘组织DNA进行测序后，对在所述α-珠蛋白基因区域内，胎儿具有的特异的单分子甲基化状态进行分析。如图2所示，在基因型为αα/αα、-- SEA/αα的胎盘DNA中，所述特异单分子甲基化状态的DNA能够被检测到，而在α-珠蛋白基因缺失后(-- SEA/-- SEA基因型)，在所述基因型的胎盘组织DNA中则不能检测到所述特异单分子甲基化状态的DNA。因此，所属特异单分子甲基化状态的DNA片段可以用于确定胎盘组织的特异来源。 In one embodiment, as shown in FIG. 1, in the genomic region where the α-globin is located, the methylation level of a single base site has extremely high noise and is not suitable for single molecule detection. However, through the analysis of single-molecule methylation pattern, it can be found that the DNA derived from placental tissue is very different from the single-molecule methylation pattern of plasma-free DNA in non-pregnant women. Further, in another embodiment, a whole genome bisulfite sequencing method genotype αα / αα, - SEA / αα and - SEA / - SEA placental tissue after DNA sequencing of the Within the α-globin gene region, the specific single molecule methylation status of the fetus is analyzed. 2, the genotype αα / αα, - placental DNA SEA / αα, said monomolecular specific DNA methylation status can be detected, while in the absence of alpha] globin gene ( - SEA /-- SEA genotype), the DNA of the specific single molecule methylation state cannot be detected in the DNA of the placenta tissue of the genotype. Therefore, the DNA fragments of the specific single molecule methylation status can be used to determine the specific source of placental tissue.
胎儿特异的DNA甲基化标记物Fetal-specific DNA methylation markers
在一个实施方案中，本公开提供了一种可以用于产前诊断胎儿α-地中海贫血的胎儿特异的DNA甲基化标记物。In one embodiment, the present disclosure provides a fetal-specific DNA methylation marker that can be used for prenatal diagnosis of fetal α-thalassemia.
在一个实施方案中，所述标记物选自人类第16号染色体第190000-230000碱基对之间的单分子甲基化标记物。In one embodiment, the marker is selected from single-molecule methylation markers between 190000-230000 base pairs of human chromosome 16.
在另一个实施方案中，所述标记物为如下碱基对的甲基化水平，所述碱基对选自人类16号染色体如下位置中的任意一个碱基对：202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645, 221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535(GRch37版本基因组)或上述碱基对中的任意两者或多于任意两者的碱基对的组合。In another embodiment, the marker is a methylation level of base pairs selected from any one of the following positions on human chromosome 16: 202146, 202148, 202161, 202170 ,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320 ,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185 ,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645, 221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535 (GRch37 Version genome) or any combination of two or more of the above base pairs.
在另一个实施方案中，所述标记物为如下碱基对的甲基化水平，所述碱基对选自人类16号染色体如下位置中的任意一个碱基对：229484,229499,229527,229535(GRch37版本基因组)或上述碱基对中的任意两者或多于任意两者的碱基对的组合。In another embodiment, the marker is a methylation level of base pairs selected from any one of the following positions on human chromosome 16: 229484,229499,229527,229535 (GRch37 version genome) or any combination of two or more of the above base pairs.
在另一个实施方案中，所述标记物为如下碱基对的甲基化水平，所述碱基对选自人类16号染色体如下位置中的任意一个碱基对：229484,229499,(GRch37版本基因组)或上述碱基对的组合。In another embodiment, the marker is a methylation level of base pairs selected from any one of the base pairs of human chromosome 16 in the following positions: 229484, 229499, (GRch37 version Genome) or a combination of the above base pairs.
在另一个实施方案中，所述标记物为如下碱基对的甲基化水平，所述碱基对选自人类16号染色体如下位置中的任意一个碱基对：229527,229535(GRch37版本基因组)或上述碱基对的组合。In another embodiment, the marker is a methylation level of base pairs selected from any one of the following positions in human chromosome 16 at the following positions: 229527,229535 (GRch37 version genome ) Or a combination of the above base pairs.
在另一个实施方案中，所述碱基对选自人类16号染色体如下位置中的任意一个碱基对：229484,229499,229527,229535(GRch37版本基因组)或上述碱基对中的任意两者或多于任意两者的碱基对的组合和选自如下一个或两者以上人类16号染色体如下位置的碱基对：202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535(GRch37版本基因组)的组合。In another embodiment, the base pair is selected from any one of the following positions on human chromosome 16: 229484, 229499, 229527, 229535 (GRch37 version genome) or any two of the above base pairs Or a combination of more than any two base pairs and base pairs selected from one or more of the following positions on human chromosome 16: 202146, 202148, 202161, 202170, 202178, 202310, 202425, 205180, 205229, 205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575, 210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243, 216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535 (GRch37 version of the genome) combination.
α-地中海贫血的无创产前检测方法Non-invasive prenatal testing method for α-thalassemia
本公开提供了一种非侵入性的胎儿α-地中海贫血产前诊断的方法，所述方法利用胎儿特异的单分子甲基化状态区分孕妇血液中的胎儿DNA和母体DNA，随后对所述胎儿DNA的拷贝数进行统计，以评估胎儿α-珠蛋白基因的基因型。The present disclosure provides a non-invasive method for prenatal diagnosis of fetal α-thalassemia, which uses fetal-specific single-molecule methylation status to distinguish fetal DNA from maternal blood in maternal DNA, and then to the fetus The copy number of DNA was counted to assess the genotype of the fetal α-globin gene.
在一个实施方案中，本公开涉及的α-地中海贫血的无创产前检测方法可以包括如下步骤：检测生物样本中DNA的甲基化模式；区分检测步骤中得到的生物样本中总DNA片段的DNA甲基化的不同模式；计算得到α-珠蛋白基因区域内胎盘组织相比于未孕女性血浆特异的DNA的甲基化模式；统计孕妇血浆DNA在α-珠蛋白基因区域以及对照区域内具有胎儿特异甲基化模式的DNA片段数占该区域内总DNA片段数的比例；比较α-珠蛋白基因区域以及对照区域内具有胎儿特异甲基化模式的DNA片段数占该区域内总DNA片段数的比例。在优选的实施方案中，通过判断前述比例的异同，来检测胎儿是否患有α-地中海贫血。In one embodiment, the non-invasive prenatal detection method for α-thalassemia according to the present disclosure may include the steps of: detecting the methylation pattern of DNA in the biological sample; distinguishing the DNA of the total DNA fragments in the biological sample obtained in the detection step Different patterns of methylation; calculated methylation patterns of placental tissue within the α-globin gene region compared to DNA specific to the plasma of non-pregnant women; statistics of maternal plasma DNA in the α-globin gene region and control region The ratio of the number of DNA fragments in fetal-specific methylation patterns to the total number of DNA fragments in this region; compare the number of DNA fragments with fetal-specific methylation patterns in the α-globin gene region and the control region to the total DNA fragments in this region The ratio of the number. In a preferred embodiment, whether the fetus has α-thalassemia is determined by judging the similarities and differences of the aforementioned ratios.
在另一个实施方案中，本公开涉及的α-地中海贫血的无创产前检测方法可以包括如下步骤：孕妇外周血中游离DNA的提取；使用DNA甲基化状态显示剂处理样本；检测处理后的样本中DNA的甲基化模式；统计具有胎儿特异甲基化模式的DNA数目；分析前述统计步骤中比对到α-珠蛋白基因区域内以及对照区域的DNA；将α-珠蛋白基因区域内胎儿DNA分数与标准对照进行比较，判断胎儿α-珠蛋白基因的基因型。In another embodiment, the non-invasive prenatal detection method of α-thalassemia involved in the present disclosure may include the steps of: extraction of free DNA from peripheral blood of pregnant women; treatment of samples using DNA methylation status indicator; detection of post-treatment The methylation pattern of DNA in the sample; count the number of DNAs with fetal-specific methylation patterns; analyze the DNA in the α-globin gene region and the control region compared in the previous statistical step; convert the α-globin gene region The fetal DNA fraction is compared with the standard control to determine the genotype of the fetal α-globin gene.
孕妇外周血中游离DNA的提取Extraction of free DNA from peripheral blood of pregnant women
从血浆中提取DNA的方法是本领域技术人员公知。可以按照DNA制备的常规方法进行。此外，也有多种商品化试剂盒提供，例如Qiagen公司的柱提取试剂盒、Life Technologies公司的磁珠法提取试剂盒等。在一些实施方案中，使用Qiagen柱提取试剂盒提取血浆样本中的DNA。在另一些实施方案中，可以不对血浆样本中的DNA进行提取，例如，使用亚硫酸氢盐直接对血清样本进行转化，随后进行DNA甲基化状态检测。Methods for extracting DNA from plasma are well known to those skilled in the art. It can be carried out in accordance with conventional methods for DNA preparation. In addition, there are also a variety of commercial kits available, such as Qiagen's column extraction kit, Life Technologies' magnetic bead extraction kit, and so on. In some embodiments, the DNA in the plasma sample is extracted using Qiagen column extraction kit. In other embodiments, the DNA in the plasma sample may not be extracted, for example, a bisulfite salt is used to directly convert the serum sample, and then the DNA methylation status is detected.
使用DNA甲基化状态显示剂处理样本Treat samples with DNA methylation status indicator
无论游离DNA提取与否，优先选择DNA甲基化状态显示剂处理样本，例如亚硫酸氢盐。亚硫酸氢盐能够将未甲基化胞嘧啶转化为尿嘧啶，而甲基化的胞嘧啶则保持不变，随后利用聚合酶链式反应对转化后DNA进行扩增，尿嘧啶则转化为胸腺嘧啶(Frommer et al.ProcNatlAcadSci USA,1992,89:1827-31)，随后检测胞嘧啶是否转化为胸腺嘧啶从而判断所述胞嘧啶是否被甲基化。所述DNA甲基化状态显示剂还包括，甲基化敏感的内切酶、识别DNA甲基化状态的抗体或者结合蛋白、具有DNA氧化剂脱氨基作用的酶类等。在另一些实施方式中，也可以不使用DNA甲基化状态显示剂处理样本，例如采用基于纳米孔或零模波导的单分子测序系统直接检测DNA分子的甲基化状态。Regardless of whether free DNA is extracted or not, DNA methylation status display agents are preferred, such as bisulfite. Bisulfite can convert unmethylated cytosine to uracil, while methylated cytosine remains unchanged, and then use polymerase chain reaction to amplify the converted DNA, and uracil to thymus Pyrimidine (Frommer et al. Proc Natl Acad Sci USA, 1992, 89: 1827-31), and then detect whether cytosine is converted into thymine to determine whether the cytosine is methylated. The DNA methylation state display agent also includes methylation-sensitive endonucleases, antibodies or binding proteins that recognize the DNA methylation state, enzymes with DNA oxidant deamination, and the like. In other embodiments, the sample may not be treated with a DNA methylation status indicator, for example, a single-molecule sequencing system based on nanopores or a zero-mode waveguide may be used to directly detect the methylation status of DNA molecules.
检测处理后的样本中DNA的甲基化模式Detection of DNA methylation patterns in processed samples
目前，有多种方法可用于DNA甲基化模式的检测。在一个技术方案中，可以使用包括高通量测序、实时PCR、数字PCR、质谱分析和微阵列等方法，用于DNA甲基化模式的检测。Currently, there are multiple methods available for the detection of DNA methylation patterns. In one technical solution, methods including high-throughput sequencing, real-time PCR, digital PCR, mass spectrometry, and microarrays can be used for detection of DNA methylation patterns.
基于本公开提供的胎儿特异的DNA标记物，上述方法均可用于胎儿α-地中海贫血产前诊断。Based on the fetal-specific DNA markers provided by the present disclosure, the above methods can be used for prenatal diagnosis of fetal α-thalassemia.
本公开可以基于高通量测序平台实施，例如使用illumina公司的Miseq、Hiseq、Nextseq和Novaseq等测序平台，Life Technologies公司的Ion Torrent或Ion Proton等测序平台，Pacific Biosciences system公司的PacBio RS II等测序平台，Oxford Nanopore Technologies公司GridIONX5等测序平台等。This disclosure can be implemented based on high-throughput sequencing platforms, such as sequencing platforms such as Miseq, Hiseq, Nextseq, and Novaseq of Illumin, sequencing platforms such as Ion Torrent or Ion Proton of Life Technologies, and PacBio RS II of Pacific Biosciences system, etc. Platform, Oxford, Nanopore Technologies GridIONX5 and other sequencing platforms.
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对样本DNA进行亚硫酸氢盐转化。A methylation detection method based on the principle of bisulfite conversion has become a conventional technique for detecting DNA methylation status, and is also well known to those skilled in the art. In a specific embodiment of the present disclosure, EZ DNA DNA-Methylation-Gold Bisulfite Conversion Kit from Zymo Research is used to perform bisulfite conversion on the sample DNA.
在本公开的一个具体实施方案中，将所述孕妇血浆DNA进行修复、加A，然后与Illumina甲基化衔接子连接，接着对上述DNA与衔接子的连接产物进行亚硫酸氢盐转化随后利用聚合酶链式反应(PCR)进行扩增，获得测序文 库，将所述测序文库在Illumina Nextseq500测序仪上进行测序，获得所述孕妇血浆DNA在全基因组范围内的甲基化状态的检测结果。In a specific embodiment of the present disclosure, the maternal plasma DNA is repaired, added with A, and then ligated with the Illumina methylated adaptor, and then the bisulfite conversion is performed on the ligation product of the DNA and the adaptor and then utilized Polymerase chain reaction (PCR) was used to amplify to obtain a sequencing library, and the sequencing library was sequenced on an Illumina Nextseq 500 sequencer to obtain a genome-wide detection result of the maternal plasma DNA.
在一些实施方案中，对孕妇血浆DNA先进行亚硫酸氢盐处理然后再构建测序文库，随后利用高通量测序平台进行检测。目前公开的多种方法和商业化试剂盒都可以对亚硫酸氢盐转化后的DNA样本构建测序文库，例如，基于随机引物扩增的方法(Fumihito et al.,Nucleic Acids Research,2012,40(17):e136-e136；Khanna et al.,Nature Methods,2013,10(10))、基于单链核苷酸与衔接子连接的方法(Raine et al.,Nucleic Acids Research,2016,45(6):e36；Gansauge et al.,Nucleic Acids Research,2017,45(10):e79)、基于末端脱氧核苷酸转移酶(Terminal deoxynucleotidyltransferase,TdT)的方法(Peng et al.,Nucleic Acids Research,2015,43(6):e35)等。在一些实施方案中，对亚硫酸氢盐转化后的DNA样本采用商业化试剂盒构建测序文库，所述商业化试剂盒可以是Illumina公司的Truseq DNA methylation试剂盒，Swift Biosciences公司的Accel-NGS Methyl-Seq DNA Library试剂盒等。In some embodiments, maternal plasma DNA is first treated with bisulfite and then a sequencing library is constructed, and then detected using a high-throughput sequencing platform. A variety of currently disclosed methods and commercial kits can construct sequencing libraries for bisulfite-converted DNA samples, for example, random primer-based amplification methods (Fumihito et al., Nucleic Acids Research, 2012, 40( 17): e136-e136; Khanna et al., Nature Methods, 2013, 10(10)), a method based on the connection of single-stranded nucleotides and adaptors (Raine et al., Nucleic Acids Research, 2016, 45(6 ): e36; Gansauge et al., Nucleic Acids Research, 2017, 45(10): e79), a method based on terminal deoxynucleotidyl transferase (TdT) (Peng et al., Nucleic Acids Research, 2015 , 43(6): e35) etc. In some embodiments, a sequencing kit is used to construct a sequencing library for the bisulfite-converted DNA sample. The commercial kit may be Illumina's Truseq DNA methylation kit, Swift Biosciences' Accel-NGS Methyl -Seq DNA Library Kit etc.
在另一些实施方案中，使用液相或固相探针对相关区域进行杂交捕获，随后进行测序，完成对目标区域DNA甲基化状态的靶向检测。一方面，所述探针可以是与基因组序列互补配对，例如将所述样本DNA进行修复、加A，然后与Illumina甲基化衔接子连接，随后将上述DNA与衔接子的连接产物与所述探针孵育杂交，清洗探针后进行亚硫酸氢盐转化随后利用聚合酶链式反应(PCR)进行扩增，获得测序文库，将所述测序文库在Illumina测序仪上进行测序，获得所述样本DNA在目的区域范围内的甲基化状态的检测结果(Hing B,Ramos et al.,Epigenetics 2015；10:581-96)。另一方面，所述探针可以是与亚硫酸氢盐转化后的DNA序列互补配对，例如将所述样本DNA进行修复、加A，然后与Illumina甲基化衔接子连接，接着进行亚硫酸氢盐转化，随后将上述扩增产物与所述探针孵育杂交，清洗探针后利用聚合酶链式反应(PCR)进行扩增获得测序文库，将所述测序文库在Illumina测序仪上进行测序，获得所述样本DNA在目的区域范围内的甲基化状态的检测结果(Allum et  al.,Nat Commun,2015；6:7211；Li et al.,Nucleic Acids Res 2015；43:e81)。在一些实施方案中，探针可以是固相的(Okou et al.,Nature Methods,2007,4(11):907；Sandoval et al.,Epigenetics,2011,6(6),692–702)。在其他一些实施方案中，与探针杂交的DNA可以是已经与衔接子连接的(Wang et al.,Bmc Genomics,2011,12(1):597)。在一些实施方案中，与探针杂交的DNA可以是未与衔接子连接的。例如，将所述DNA与RNA探针杂交孵育，随后对探针进行清洗，接着使用RNA酶降解RNA探针，随后对捕获的DNA进行文库构建后进行亚硫酸氢盐处理，接着进行PCR扩增(或对捕获的DNA进行亚硫酸氢盐处理后再进行文库构建，接着进行PCR扩增)，最后对测序文库进行测序。In other embodiments, a liquid phase or solid phase probe is used to hybridize and capture the relevant regions, followed by sequencing to complete the targeted detection of the DNA methylation status of the target region. On the one hand, the probe may be complementary to the genomic sequence, for example, the sample DNA is repaired, A is added, and then the Illumina methylated adaptor is ligated, and then the ligation product of the DNA and the adaptor is connected to the The probe is incubated for hybridization, the probe is washed, bisulfite conversion is performed, and then amplified by polymerase chain reaction (PCR) to obtain a sequencing library, and the sequencing library is sequenced on an Illumina sequencer to obtain the sample Detection results of DNA methylation status within the target area (Hing B, Ramos et al., Epigenetics 2015; 10:581-96). On the other hand, the probe may be complementary paired with the bisulfite converted DNA sequence, for example, the sample DNA is repaired, A is added, and then connected to the Illumina methylated adaptor, followed by bisulfite Salt transformation, and then incubating and hybridizing the above amplification product with the probe, washing the probe and performing amplification using polymerase chain reaction (PCR) to obtain a sequencing library, and sequencing the sequencing library on an Illumina sequencer, Obtain the detection results of the methylation status of the sample DNA within the target area (Allum et al., Nat Commun, 2015; 6:7211; Li et al., Nucleic Acids Res. 2015; 43:e81). In some embodiments, the probe may be solid phase (Okou et al., Nature Methods, 2007, 4(11): 907; Sandoval et al., Epigenetics, 2011, 6(6), 692-702). In some other embodiments, the DNA that hybridizes to the probe may be already ligated to the adaptor (Wang et al., Bmc Genomics, 2011, 12(1):597). In some embodiments, the DNA that hybridizes to the probe may not be linked to the adaptor. For example, the DNA is hybridized and incubated with an RNA probe, and then the probe is washed, then the RNA probe is degraded using RNase, and then the captured DNA is subjected to bisulfite treatment after library construction, followed by PCR amplification (Or perform bisulfite treatment on the captured DNA and then construct the library, followed by PCR amplification), and finally sequence the sequencing library.
在另一些实施方案中，基于简化代表性亚硫酸氢盐测序法检测所述DNA甲基化状态(RRBS，Meissner et al.,Nucleic Acids Res,2005；33:5868–5877；Gu et al.,Nat Protoc.2011；6,(4),468-481)。在一些实施方案中，用于RRBS测序的内切酶可以根据本公开提供的胎儿特异的DNA甲基化标记物区域进行挑选。在其他一些实施方案中，基于甲基化DNA抗体或结合蛋白的免疫沉淀测序法检测所述DNA甲基化状态(MeDIP-Seq，Jacinto et al.,Biotechniques,2008；44(1),35–43；MBD-seq，Serre et al.,Nucleic Acids Res 2010；38:391–399.)。在另一些实施方案中，基于甲基化敏感限制性内切酶测序法检测所述DNA甲基化状态(MRE-seq，Maunakea et al.,Nature.2010；466:253–257)，所述甲基化敏感限制性内切酶可以根据本公开提供的胎儿特异的DNA甲基化标记物区域进行挑选。In other embodiments, the DNA methylation status is detected based on a simplified representative bisulfite sequencing method (RRBS, Meissner et al., Nucleic Acids Res, 2005; 33:5868-5877; Gu et al., Nat Protoc. 2011; 6, (4), 468-481). In some embodiments, the endonuclease used for RRBS sequencing can be selected based on the fetal-specific DNA methylation marker regions provided by the present disclosure. In some other embodiments, the DNA methylation status is detected by immunoprecipitation sequencing based on methylated DNA antibodies or binding proteins (MeDIP-Seq, Jacinto et al., Biotechniques, 2008; 44(1), 35- 43; MBD-seq, Serre et al., Nucleic Acids Res 2010; 38:391–399.). In other embodiments, the DNA methylation status is detected based on methylation-sensitive restriction enzyme sequencing (MRE-seq, Maunakea et al., Nature. 2010; 466:253-257), Methylation-sensitive restriction enzymes can be selected based on the fetal-specific DNA methylation marker regions provided by the present disclosure.
在一些实施方案中，所述样本DNA经亚硫酸氢盐处理后，利用聚合酶链式反应(PCR)扩增目的区域，随后对扩增产物进行测序，分析样本DNA的甲基化状态。在一些实施方案中，所述PCR可以是多重PCR。在一些实施方案中，利用微滴技术进行所述PCR扩增(Komori et al.,Genome Research,2011,21(10):1738-1745)。In some embodiments, after the sample DNA is treated with bisulfite, the target region is amplified by polymerase chain reaction (PCR), and then the amplified product is sequenced to analyze the methylation status of the sample DNA. In some embodiments, the PCR may be multiplex PCR. In some embodiments, the PCR amplification is performed using microdrop technology (Komori et al., Genome Research, 2011, 21(10): 1738-1745).
单分子测序平台(例如，Oxford Nanopore公司的MinION、Pacific  Biosciences system公司的PacBio RS II等测序平台)将允许在无亚硫酸氢盐转化下直接检测DNA分子的甲基化状态(包括N6-甲基腺嘌呤、5-甲基胞嘧啶和5-羟甲基胞嘧啶(Flusberg et al.,7:461-465Nature methods；J Shim 2013Sci Rep 3:1389))。在一些实施方案中，不经亚硫酸氢盐转化，采用单分子测序平台分析的样本DNA的甲基化状态。Single-molecule sequencing platforms (e.g., Oxford Nanopore’s MinION, Pacific Biosciences system’s PacBio RS II and other sequencing platforms) will allow direct detection of the methylation status of DNA molecules (including N6-methyl) without bisulfite conversion Adenine, 5-methylcytosine and 5-hydroxymethylcytosine (Flusberg et al., 7:461-465 Nature methods; J Shim 2013 Sci Rep 3:1389)). In some embodiments, the methylation status of sample DNA analyzed using a single molecule sequencing platform without bisulfite conversion.
在另一些实施方案中，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，基于本公开提供的胎儿特异的DNA甲基化标记物，使用任何可以检测DNA甲基化状态的方法均可实现胎儿α-地中海贫血的产前诊断，包括但不限于胎儿的非侵入性产前诊断。In other embodiments, other techniques can be used to analyze the DNA methylation status, such as microarray, real-time PCR, digital real-time PCR, and mass spectrometry analysis (Plongthongkum et al., Nature Reviews Genetics, 2014, 15(10) :647-61; Consortium et al., Nature Biotechnology, 2016, 34(7):726). Those skilled in the art should readily recognize that based on the fetal-specific DNA methylation markers provided by the present disclosure, any method that can detect the DNA methylation status can achieve prenatal diagnosis of fetal α-thalassemia, including but Not limited to non-invasive prenatal diagnosis of the fetus.
DNA甲基化模式的分析Analysis of DNA methylation patterns
“甲基化模式”是对样本DNA在所述区域内整体甲基化状态的描述。在一些实施方案中，DNA甲基化模式的分析可以选自DNA甲基化状态的分析。"Methylation mode" is a description of the overall methylation status of the sample DNA in the region. In some embodiments, the analysis of DNA methylation patterns may be selected from the analysis of DNA methylation status.
在一些实施方案中，所述“DNA甲基化模式的分析”为统计具有胎儿特异甲基化模式的DNA数目。In some embodiments, the "analysis of DNA methylation patterns" is to count the number of DNAs with fetal-specific methylation patterns.
在一些实施方案中，经亚硫酸氢盐处理后，建库、测序所得到的DNA片段信息，可以使用领域内人员公知的方法，如，bismark(Felix Kruegerand Simon R.Andrews(2011).Bismark:a flexible aligner and methylation caller for Bisulfite-Seq applications.Bioinformatics)，bwa-meth(Brent S.Pedersen,et al.(2014).Fast and accurate alignment of long bisulfite-seq reads.Bioinformatics)等方法，将DNA片段比对到人类参考基因组，如：GRCh37(Feb.2009,hg19)。在此过程中，参考基因组上为C碱基、测序片段上为T碱基的错配将被认为是未甲基化的C碱基，而未发生错配的C碱基则会被认为是甲基化的C碱基。每一条单分子DNA片段上的碱基会被分析，用于定义其携带的单分子甲基化模式。进一步地，具有特定甲基化模式的DNA片段， 被归类为不同的来源。该归类方法是根据前述的单分子甲基化模式进行的。比对后得到bam文件，使用biscuit工具包(version:0.2.0.20161222)的epiread命令，提取DNA片段的单分子甲基化模式。通过R语言的统计软件包MASS(version：7.3.51.1)分析，获得胎盘基因组相比于未孕女性游离DNA基因组，稳定存在的特异的单分子甲基化模式。In some embodiments, after the bisulfite treatment, the DNA fragment information obtained by library construction and sequencing can use methods well known to those in the field, such as bismark (Felix Kruegerand Simon R. Andrews (2011). Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications.Bioinformatics), bwa-meth (Brent S. Pedersen, et Al. (2014). Fast and accurate alignment of long bisulfite-seq reads. Bioinformatics) and other methods to convert DNA fragments Compare to the human reference genome, such as: GRCh37 (Feb.2009, hg19). In this process, a mismatch of C bases on the reference genome and T bases on the sequenced fragments will be considered unmethylated C bases, and C bases that have not been mismatched will be considered as Methylated C base. The bases on each single-molecule DNA fragment are analyzed to define the single-molecule methylation pattern it carries. Further, DNA fragments with specific methylation patterns are classified as different sources. The classification method is based on the aforementioned single molecule methylation mode. After the comparison, the bam file is obtained, and the epiread command of the biscuit toolkit (version:0.2.0.20161222) is used to extract the single molecule methylation pattern of the DNA fragment. According to the analysis of the statistical software package MASS (version: 7.3.51.1) in R language, a specific single-molecule methylation pattern stably existing in the placental genome compared to the free DNA genome of infertile women was obtained.
在一些实施方案中，符合本公开提供的胎儿特异的DNA甲基化模式的DNA片段，可被认为是胎儿来源的DNA片段。本领域技术人员应容易认识到，基于本公开提供的胎儿特异的DNA甲基化标记物，使用任何合理的统计手段归类DNA片段，均可实现胎儿α-地中海贫血的产前诊断。In some embodiments, DNA fragments that conform to the fetal-specific DNA methylation pattern provided by the present disclosure can be considered as DNA fragments of fetal origin. Those skilled in the art should readily recognize that, based on the fetal-specific DNA methylation markers provided by the present disclosure, using any reasonable statistical means to classify the DNA fragments, the prenatal diagnosis of fetal α-thalassemia can be achieved.
在一些实施方案中，胎儿α-地中海贫血的产前诊断可以选自胎儿的非侵入性产前诊断。In some embodiments, the prenatal diagnosis of fetal α-thalassemia can be selected from non-invasive prenatal diagnosis of the fetus.
判断胎儿α-珠蛋白基因的基因型Determine the genotype of fetal α-globin gene
在一些实施方案中，所述“判断胎儿α-珠蛋白基因的基因型”可以为将DNA甲基化模式的分析步骤中的结果，例如统计具有胎儿特异甲基化模式的DNA数目，比对到α-珠蛋白基因区域内以及对照区域的DNA。将α-珠蛋白基因区域内胎儿DNA分数与标准对照进行比较，判断胎儿α-珠蛋白基因的基因型。In some embodiments, the "judgment of the genotype of the fetal α-globin gene" may be the result of the analysis step of the DNA methylation pattern, such as counting the number of DNAs with fetal specific methylation pattern, and comparing DNA into the α-globin gene region and the control region. The fetal DNA fraction in the α-globin gene region is compared with the standard control to determine the genotype of the fetal α-globin gene.
在一些实施方案中，根据DNA甲基化模式的分析步骤中的分析结果，可统计在对照基因组区域(非α-珠蛋白基因组区域)和α-珠蛋白基因组区域上，胎儿来源的DNA片段的数目。进而，计算出在某一基因组区域上，胎儿来源DNA片段占该区域上所有DNA片段的比例(下称“局部胎源比例”)。在一些实施方案中，局部胎源比例是由一个单分子甲基化状态所定义的。在另一些实施方案中，局部胎源比例可以由两个或更多单分子甲基化状态所定义。在一些实施方案中，局部胎源比例是单分子甲基化状态所对应胎源比例的平均值，在另一些实施方案中，局部胎源比例是单分子甲基化状态所对应胎源比例的加权平均值。由于对照基因组区域被默认为总是正常的，因此，比较α-珠蛋白基因组区域的局部胎源比例和对照基因组区域的局部胎源比 例之比(下称，相对α-珠蛋白基因比例)，即可推测胎儿α-珠蛋白基因组区域的拷贝数。在一些实施方案中，只需统计目标被测试孕妇外周血的相对α-珠蛋白基因比例，便可推测其携带胎儿的基因型。在另一些实施方案中，还可以从携带已知α-珠蛋白基因型胎儿的孕妇外周血游离DNA进行测序，并统计其相对α-珠蛋白基因比例，并以此计算统计分布，进而，利用数学统计检验，推测目标被测试孕妇携带胎儿的α-珠蛋白基因型。本领域技术人员应容易认识到，基于本公开提供的胎儿特异的DNA甲基化标记物，使用任何合理的统计手段，均可实现胎儿α-地中海贫血的产前诊断，包括但不限于胎儿的非侵入性产前诊断。In some embodiments, according to the analysis result in the analysis step of the DNA methylation pattern, the fetal-derived DNA fragments can be counted on the control genomic region (non-α-globin genomic region) and α-globin genomic region number. Furthermore, the ratio of the DNA fragments derived from the fetus to all the DNA fragments in the region in a certain genomic region is calculated (hereinafter referred to as "local fetal proportion"). In some embodiments, the local fetal proportion is defined by a single molecule methylation status. In other embodiments, the local fetal proportion may be defined by two or more single molecule methylation states. In some embodiments, the local fetal source ratio is the average of the single-molecule methylation state corresponding to the fetal source ratio. In other embodiments, the local fetal source ratio is the single-molecule methylation state corresponding to the fetal source ratio. Weighted average. Since the control genome region is always normal by default, the ratio of the local fetal proportion of the α-globin genomic region and the local fetal proportion of the control genomic region (hereinafter referred to as the relative α-globin gene ratio) is compared, The copy number of the fetal α-globin genomic region can be estimated. In some embodiments, it is only necessary to count the relative proportion of α-globin genes in the peripheral blood of the target pregnant woman to be able to infer the genotype of the fetus it carries. In other embodiments, the free DNA from the peripheral blood of pregnant women carrying fetuses with known α-globin genotypes can also be sequenced, and the relative α-globin gene ratios can be counted, and the statistical distribution can be calculated based on this. Mathematical statistical tests speculate that the target pregnant woman carries the fetal α-globin genotype. Those skilled in the art should readily recognize that based on the fetal-specific DNA methylation markers provided by the present disclosure, using any reasonable statistical means, the prenatal diagnosis of fetal α-thalassemia can be achieved, including but not limited to fetal Non-invasive prenatal diagnosis.
在另一个实施方案中，本公开涉及的α-地中海贫血的无创产前检测方法可以包括如下步骤：检测样本获得；检测样本的预处理。In another embodiment, the non-invasive prenatal detection method of α-thalassemia according to the present disclosure may include the following steps: obtaining a test sample; preprocessing the test sample.
检测样本获得Test samples obtained
在一个实施方案中，所述“检测样本获得”步骤为孕妇外周血的获取步骤。In one embodiment, the step of "obtaining a test sample" is a step of obtaining peripheral blood of a pregnant woman.
孕妇外周血的获取步骤包括如下步骤：采集含有母体DNA和胎儿DNA的孕妇的生物样本，所述生物学样本包括但不限于孕妇的血液、尿液和唾液等，优选孕妇的血液样本。所述血液样本包括但不限于孕妇的外周血。采集适合孕龄的孕妇外周血，所述孕龄可以为4周、6周、8周、10周、12周、14周、16周、18周、20周、22周、24周、28周、32周、36周、40周、或上述时间点的任两者之间的时间，优选6周-28周、更优选10周-22周。按照医院或机构所采用的标准流程采集、运输或保存孕妇的外周血。采集孕妇外周血的容器可以是使用商业化的产品，例如Streck Tube采血管。The step of obtaining the peripheral blood of a pregnant woman includes the following steps: collecting a biological sample of a pregnant woman containing maternal DNA and fetal DNA, the biological sample including but not limited to the blood, urine and saliva of the pregnant woman, preferably a blood sample of the pregnant woman. The blood sample includes but is not limited to the peripheral blood of pregnant women. Collect peripheral blood of pregnant women suitable for gestational age, the gestational age can be 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 28 weeks , 32 weeks, 36 weeks, 40 weeks, or any time between the above time points, preferably 6 weeks to 28 weeks, more preferably 10 weeks to 22 weeks. Collect, transport or preserve the peripheral blood of pregnant women in accordance with standard procedures adopted by hospitals or institutions. The container for collecting peripheral blood of pregnant women can be a commercial product such as Streck Tube blood collection tube.
检测样本的预处理Pretreatment of test samples
在一个实施方案中，所述“检测样本的预处理”步骤为孕妇外周血血浆的制备步骤。In one embodiment, the step of "pretreatment of the test sample" is a step of preparing blood plasma of pregnant women.
孕妇外周血血浆的制备步骤包括如下步骤：从母体外周血中分离血浆或血清的方法是本领域技术人员所公知。例如，以1600g对所述孕妇外周血样 本进行离心，获得血浆或血清。在一个具体的实施方案中，在4℃条件下以1600g对Streck Tube采血管采集的外周血样本进行离心，随后将血浆转移到新的离心管中，再在4℃条件下以16000g对血浆进行离心去除残留血细胞。The preparation steps of the peripheral blood plasma of pregnant women include the following steps: A method of separating plasma or serum from maternal peripheral blood is well known to those skilled in the art. For example, the peripheral blood sample of the pregnant woman is centrifuged at 1600 g to obtain plasma or serum. In a specific embodiment, the peripheral blood sample collected from the Streck Tube blood collection tube is centrifuged at 1600g at 4°C, and then the plasma is transferred to a new centrifuge tube, and the plasma is then subjected to 16000g at 4°C. Centrifuge to remove residual blood cells.
实施例Examples
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。Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although indicating specific embodiments of the present disclosure) are given for explanatory purposes only, because after reading this detailed description, it is made within the spirit and scope of the present disclosure Various changes and modifications will become apparent to those skilled in the art.
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。Unless otherwise emphasized, all reagents used in this example can be purchased through commercial channels.
实施例1：胎盘组织中DNA和未孕女性血浆中游离DNA的甲基化状态的Example 1: Methylation status of DNA in placental tissue and free DNA in plasma of infertile women 测定Determination
1受试者招募和样本采集1 Subject recruitment and sample collection
从广东省妇女儿童保健院产前诊断科招募受试者。本研究和人类临床样本的采集得到机构审查委员会的批准，并获得每位受试者的知情同意书。在选择性妊娠终止后立即采集妊娠早期的胎盘组织。在单纯性妊娠的选择性剖腹产后立即采集妊娠晚期的胎盘组织。采集未孕女性志愿者的外周血(10mL，Streck 218962 10mL Cell-Free DNA BCT无创采血管)。Subjects were recruited from the Prenatal Diagnosis Department of Guangdong Women and Children's Health Hospital. The study and the collection of human clinical samples were approved by the institutional review board, and each subject’s informed consent was obtained. Immediately after the termination of selective pregnancy, placental tissues in the first trimester were collected. Immediately after the selective cesarean section of simple pregnancy, the placental tissue of the third trimester was collected. Collect peripheral blood (10 mL, Streck 218962, 10 mL Cell-Free DNA, BCT non-invasive blood collection tube) of infertile female volunteers.
2样本处理2 Sample processing
将外周血样本于4℃条件下以1600g离心10分钟，将血浆部分再于4℃条件下以16000g离心10分钟，进一步去除残留血细胞。The peripheral blood sample was centrifuged at 1600g for 10 minutes at 4°C, and the plasma fraction was centrifuged at 16000g for 10 minutes at 4°C to further remove residual blood cells.
3 DNA提取3 DNA extraction
按照制造商的说明书，利用DNeasy Blood&Tissue试剂盒(制造商：QIAGEN；目录号：69506)从胎盘组织中提取DNA。按照制造商的说明书，利用QIAamp Circulating Nucleic Acid提取试剂盒(制造商：QIAGEN；目录号：55114)从血浆中提取游离DNA。According to the manufacturer's instructions, DNA was extracted from the placental tissue using the DNeasy Blood & Tissue kit (manufacturer: QIAGEN; catalog number: 69506). According to the manufacturer's instructions, free DNA was extracted from the plasma using QIAamp Circulating Nucleic Acid Extraction Kit (manufacturer: QIAGEN; catalog number: 55114).
4全基因组亚硫酸氢盐测序(WGBS)4 Whole Genome Bisulfite Sequencing (WGBS)
4.1胎盘组织DNA片段化处理4.1 DNA fragmentation of placenta tissue
利用S220型超声波破碎仪(制造商：Covaris；目录号：S220)，按照表1所示参数，将胎盘组织DNA破碎，达到片段化目的。Using the S220 ultrasonic breaker (manufacturer: Covaris; catalog number: S220), according to the parameters shown in Table 1, the DNA of the placenta tissue was fragmented to achieve the purpose of fragmentation.
表1胎盘组织DNA片段化处理参数Table 1 DNA fragmentation processing parameters of placental tissue
Figure PCTCN2019123983-appb-000001
Figure PCTCN2019123983-appb-000001
4.2 DNA与甲基化衔接子连接4.2 DNA and methylated adaptor connection
利用HyperPreP建库试剂盒，将1μg已片段化的胎盘组织DNA或10-50ng非孕女性游离DNA进行修复、加A，随后连接Illumina甲基化Truseq衔接子(引物序列号；001/002)。前述步骤中采用的试剂或试剂盒的来源如表2A所示。前述步骤中采用的试剂或试剂盒的来源如表2B所示。Using the HyperPreP library construction kit, 1 μg of fragmented placental tissue DNA or 10-50 ng of non-pregnant female free DNA was repaired, A was added, and then an Illumina methylated Truseq adaptor (primer sequence number; 001/002) was connected. The sources of the reagents or kits used in the foregoing steps are shown in Table 2A. The sources of the reagents or kits used in the foregoing steps are shown in Table 2B.
表2A采用的引物的序列Table 2A Sequence of primers used
Figure PCTCN2019123983-appb-000002
Figure PCTCN2019123983-appb-000002
表2B步骤1.4.2中试剂或试剂盒的来源Table 2B Source of reagents or kits in step 1.4.2
Figure PCTCN2019123983-appb-000003
Figure PCTCN2019123983-appb-000003
DNA修复和纯化DNA repair and purification
按照表3所示配制修复反应液，充分混匀后，于20℃条件下反应30分钟。Prepare the repair reaction solution as shown in Table 3, mix thoroughly, and react at 20°C for 30 minutes.
表3修复反应液的组份及含量Table 3 Composition and content of repair reaction solution
反应组分Reaction component 体积(μL)Volume (μL)
DNA DNA 5050
Nuclease-free WaterNuclease-free Water 88
10X KAPA End Repair Buffer10X KAPA End Repair Buffer 77
KAPA End Repair Enzyme MixKAPAEndRepairEnzymeMix 55
反应结束后，加入120μL已室温孵育30分钟的AgencourtAMPure XP磁珠，纯化DNA。按照HyperPreP建库试剂盒的说明书，不将DNA从磁珠上洗脱。After the reaction, 120 μL of AgencourtAMPure XP magnetic beads incubated at room temperature for 30 minutes were added to purify the DNA. According to the instructions of the HyperPreP library building kit, DNA is not eluted from the magnetic beads.
DNA的A-Tailing和纯化A-Tailing and purification of DNA
按照表4所示配制DNA的A-Tailing反应液，充分混匀后，于30℃条件下反应30分钟。Prepare the A-Tailing reaction solution of DNA as shown in Table 4. After thoroughly mixing, react at 30°C for 30 minutes.
表4 DNA的A-Tailing反应液配制Table 4 Preparation of DNA A-Tailing reaction solution
反应组分Reaction component 体积(μL)Volume (μL)
AgencourtAMPure XP磁珠(DNA)AgencourtAMPure XP magnetic beads (DNA) --
Nuclease-free WaterNuclease-free Water 4242
10X KAPA A-Tailing Buffer10X KAPA A-Tailing Buffer 55
KAPA A-Tailing EnzymeKAPA A-Tailing Enzyme 33
反应结束后，加入90μL已室温孵育30分钟的PEG/NaCl
Figure PCTCN2019123983-appb-000004
Solution磁珠，纯化DNA，得到末端加A的DNA。 After the reaction, add 90 μL of PEG/NaCl that has been incubated at room temperature for 30 minutes
Figure PCTCN2019123983-appb-000004
Solution magnetic beads to purify DNA to obtain DNA with A added at the end.
DNA与衔接子连接DNA to adaptor
按照表5所示配制连接反应液，充分混匀后，于20℃条件下反应15分钟。The ligation reaction solution was prepared as shown in Table 5, mixed thoroughly, and reacted at 20°C for 15 minutes.
表5连接反应液配制Table 5 Preparation of connection reaction solution
反应组分Reaction component 体积(μL)Volume (μL)
AgencourtAMPure XP磁珠(DNA)AgencourtAMPure XP magnetic beads (DNA) --
Nuclease-free WaterNuclease-free Water 3030
5X KAPA Ligation Buffer 5X KAPALigationBuffer 1010
衔接子(10μM，001/002)Adaptor (10μM, 001/002) 55
KAPA DNA LigaseKAPADNALigase 55
反应结束后，加入50μL已室温孵育30分钟的PEG/NaCl
Figure PCTCN2019123983-appb-000005
Solution磁珠，纯化DNA，最后加入20μL Nuclease-free Water洗脱，得到与衔接子连接的DNA。 After the reaction, add 50 μL of PEG/NaCl that has been incubated at room temperature for 30 minutes
Figure PCTCN2019123983-appb-000005
Solution magnetic beads, purify the DNA, and finally add 20 μL Nuclease-free Water to elute to obtain the DNA linked to the adaptor.
4.3亚硫酸氢盐转化4.3 Bisulfite conversion
按照制造商的说明书，利用EZ DNA Methylation-Gold亚硫酸氢盐转化试剂盒(制造商：ZYMO Research；目录号：D5005)对已连接衔接子的DNA进行亚硫酸氢盐转化，最后使用23μL Nuclease-free Water洗脱。According to the manufacturer's instructions, use the EZ DNA Methylation-Gold Bisulfite Conversion Kit (manufacturer: ZYMO Research; catalog number: D5005) to perform bisulfite conversion on the DNA of the connected adaptor, and finally use 23 μL Nuclease- free Water elution.
4.4 PCR扩增和测序文库纯化4.4 PCR amplification and sequencing library purification
按照制造商的说明书，利用KAPA HiFiHotStart Uracil+ReadyMix PCR扩增试剂盒(制造商：Roche KapaBiosystems；目录号：KK2802)对亚硫酸氢盐转化的DNA进行PCR扩增。PCR反应结束后，使用AgencourtAMPure XP磁珠纯化DNA，获得测序文库。According to the manufacturer's instructions, KAPA HiFi Hot Start Uracil + Ready Mix PCR amplification kit (manufacturer: Roche Kapa Biosystems; catalog number: KK2802) was used for PCR amplification of bisulfite-converted DNA. After the PCR reaction was completed, DNA was purified using AgencourtAMPure XP magnetic beads to obtain a sequencing library.
4.5测序文库定量及测序4.5 Quantification and sequencing of sequencing libraries
按照制造商的说明书，利用安捷伦2100生物分析仪(Agilent Technologies，目录号G2939BA)和安捷伦高灵敏DNA试剂盒(Agilent Technologies，目录号5067-4626)检测文库片段分布；按照制造商的说明书，利用文库定量试剂盒(KAPA Biosystems，目录号KK4824)以及DNA定量标准品和预混引物试剂盒(KAPA Biosystems，目录号KK4808)检测测序文库浓度。Use the Agilent 2100 Bioanalyzer (Agilent Technologies, catalog number G2939BA) and Agilent High Sensitive DNA Kit (Agilent Technologies, catalog number 5067-4626) to detect the distribution of library fragments according to the manufacturer's instructions; use the library according to the manufacturer's instructions Quantitative kits (KAPA Biosystems, catalog number KK4824) and DNA quantification standards and premix primer kits (KAPA Biosystems, catalog number KK4808) were used to detect sequencing library concentrations.
按照制造商的说明书，利用IlluminaNextseq测序仪对测序文库进行测序。According to the manufacturer's instructions, the sequencing library was sequenced using the IlluminaNextseq sequencer.
实施例2：胎儿特异的甲基化模式标志物的鉴定Example 2: Identification of fetal-specific methylation pattern markers
先前的研究表明，胎儿游离DNA主要来自于胎盘组织(Papageorgiou et al.,Am J Pathol 2009；174:1609–1618；Lun et al.,ClinChem 2013；59:1583–1594)，因此，我们分析了胎盘组织基因组的甲基化模式，同时使用非孕女性血浆游离DNA作为参考，以鉴定胎儿特异的甲基化模式标志物。Previous studies have shown that fetal free DNA mainly comes from placental tissue (Papageorgiou et al., Am J Pathol 2009; 174: 1609–1618; Lun et al., ClinChem 2013; 59: 1583–1594), therefore, we analyzed The methylation pattern of the placental tissue genome, while using plasma free DNA from non-pregnant women as a reference, to identify fetal-specific methylation pattern markers.
基于实施例1得到的测序结果，进行进一步的数据分析。使用bwa-meth(Version:0.2.0)对测序后数据进行比对。比对后结果，通过使用biscuit工具包(version:0.2.0.20161222)的epiread命令，提取DNA片段的单分子甲基化模式。通过R语言的统计软件包MASS(version：7.3.51.1)分析，获得胎盘基因组相比于未孕女性游离DNA基因组，稳定存在的特异的单分子甲基化模式。Based on the sequencing results obtained in Example 1, further data analysis was performed. Use bwa-meth(Version:0.2.0) to compare the sequenced data. After the comparison, the single molecule methylation pattern of the DNA fragment was extracted by using the epiread command of the biscuit toolkit (version:0.2.0.20161222). According to the analysis of the statistical software package MASS (version: 7.3.51.1) in R language, a specific single-molecule methylation pattern stably existing in the placental genome compared to the free DNA genome of infertile women was obtained.
实验结果：在基因组(GRCh37版本)上，寻找出全基因组上所存在的所有可能的胎儿特异甲基化位点，如图1A-图1C所示。这些基因组位点，在胎儿中与母体中，甲基化水平具有很大差异。Experimental results: On the genome (GRCh37 version), find all possible fetal-specific methylation sites present on the whole genome, as shown in FIGS. 1A-1C. These genomic loci have very different levels of methylation in the fetus and the mother.
实施例3：胎盘绒毛组织中DNA和未孕女性血浆中游离DNA的甲基化状Example 3: Methylation of DNA in placental villi and free DNA in plasma of infertile women 态的测定State determination
1.受试者招募和样本采集1. Subject recruitment and sample collection
从广州市妇女儿童健康中心产前诊断科招募受试者。本研究和人类临床样本的采集得到机构审查委员会的批准，并获得每位受试者的知情同意书。在孕中期(11-14周孕龄)在超声引导下采集胎盘绒毛。采集未孕女性志愿者的外周血(10mL，Streck 218962 10mL Cell-Free DNA BCT无创采血管)。Subjects were recruited from the Prenatal Diagnosis Department of Guangzhou Women and Children Health Center. The study and the collection of human clinical samples were approved by the institutional review board, and each subject’s informed consent was obtained. In the second trimester (11-14 weeks gestational age), placental villi were collected under ultrasound guidance. Collect peripheral blood (10 mL, Streck 218962, 10 mL Cell-Free DNA, BCT non-invasive blood collection tube) of infertile female volunteers.
2.样本处理2. Sample processing
将外周血样本于室温条件下以1600g离心10分钟，将血浆部分再于室温条件下以16000g离心10分钟，进一步去除残留血细胞。。The peripheral blood sample was centrifuged at 1600g for 10 minutes at room temperature, and the plasma fraction was centrifuged at 16000g for 10 minutes at room temperature to further remove residual blood cells. .
3.DNA提取3. DNA extraction
按照制造商的说明书，利用DNeasy Blood&Tissue试剂盒(制造商：QIAGEN；目录号：69506)从胎盘绒毛组织中提取DNA。按照制造商的说明书，利用QIAamp Circulating Nucleic Acid提取试剂盒(制造商：QIAGEN；目录号：55114)从血浆中提取游离DNA。According to the manufacturer's instructions, DNA was extracted from placental villi tissue using DNeasy Blood & Tissue kit (manufacturer: QIAGEN; catalog number: 69506). According to the manufacturer's instructions, free DNA was extracted from the plasma using QIAamp Circulating Nucleic Acid Extraction Kit (manufacturer: QIAGEN; catalog number: 55114).
4.全基因组亚硫酸氢盐测序(WGBS)4. Whole genome bisulfite sequencing (WGBS)
实施方法与实施例1中的相应部分相同。The implementation method is the same as the corresponding part in Example 1.
5.测序后分析：使用bwa-meth(Version:0.2.0)对测序后数据进行比对。根据实施例2.中发现的胎儿特异单分子甲基化标志物，使用samtools(Version:1.3.1){Heng Li et al.(2009).The Sequence Alignment/Map format and SAMtools.Bioinformatics}提取所有覆盖胎儿特异的甲基化标志物的DNA片段。使用biscuit工具包(Version:0.2.0.20161222)的epiread命令，提取上述DNA片段的单分子甲基化模式。通过R语言的统计软件包MASS(Version：7.3.51.1)分析，获得符合该模式甲基化标志物的片段。5. Post-sequencing analysis: use bwa-meth(Version:0.2.0) to compare the data after sequencing. According to the fetal-specific single-molecule methylation markers found in Example 2, use samtools (Version: 1.3.1) {Heng Li Li et al. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics} to extract all DNA fragments covering fetal-specific methylation markers. Use the epiread command of the biscuit toolkit (Version:0.2.0.20161222) to extract the single-molecule methylation pattern of the above DNA fragment. Through the analysis of the statistical software package MASS (Version: 7.3.51.1) in R language, fragments corresponding to the methylation markers of this model were obtained.
实验结果：在基因组(GRCh37版本)上，在实施例一中发现的全基因组上所存在的所有可能的胎儿特异甲基化位点附近，发现胎儿特异的单分子DNA片段甲基化模式，如图2所示(其中，图2中的A部分和图2中的B部分列出了原始测序结果，其中甲基化修饰以黑色表示，去甲基化修饰以白色表示， 其它碱基以灰色表示，上方的分布图为每个碱基上平均的甲基化修饰情况，下方的DNA片段图为每条原始测序所得片段上甲基化模式)。这些DNA片段上的甲基化模式，在胎儿中与母体中，甲基化水平具有很大差异。某些单分子甲基化模式，只存在于胎儿来源组织中，而不存在于母体中。因此，可以利用以上发现的单分子甲基化模式，精确溯源每条DNA片段的来源。Experimental results: On the genome (GRCh37 version), in the vicinity of all possible fetal-specific methylation sites found on the whole genome found in Example 1, fetal-specific single-molecule DNA fragment methylation patterns were found, such as As shown in Figure 2 (where Part A in Figure 2 and Part B in Figure 2 list the original sequencing results, the methylation modification is shown in black, the demethylation modification is shown in white, and the other bases are shown in gray Indicates that the upper distribution diagram is the average methylation modification on each base, and the lower DNA fragment diagram is the methylation pattern on each original sequenced fragment). The methylation pattern on these DNA fragments is very different in fetal and maternal levels. Some single-molecule methylation patterns only exist in tissues of fetal origin, but not in the mother. Therefore, the single-molecule methylation pattern discovered above can be used to accurately trace the source of each DNA fragment.
实施例4：α-珠蛋白基因组区域的胎儿特异的甲基化标志物分析Example 4: Analysis of fetal-specific methylation markers in the α-globin genomic region
1.绒毛基因型的判定：使用Gap-PCR法。1. Determination of villi genotype: Gap-PCR method is used.
按照制造商的说明书，利用深圳益生堂α-地中海贫血基因检测试剂盒(Gap-PCR法)(国药准字S20060084)检测胎儿绒毛的基因型。According to the manufacturer's instructions, use the Shenzhen Yishengtang α-thalassemia gene detection kit (Gap-PCR method) (National Pharmaceutical Standard S20060084) to detect the genotype of fetal villi.
2.测序后分析：使用bwa-meth(Version:0.2.0)对测序后数据进行比对。根据实施例2中发现的胎儿特异单分子甲基化标志物，使用samtools(Version:1.3.1){Heng Li et al.(2009).The Sequence Alignment/Map format and SAMtools.Bioinformatics}提取所有覆盖胎儿特异的甲基化标志物的DNA片段。使用biscuit工具包(Version:0.2.0.20161222)的epiread命令，提取上述DNA片段的单分子甲基化模式。通过R语言的统计软件包MASS(Version：7.3.51.1)分析，获得符合该模式甲基化标志物的片段。2. Analysis after sequencing: use bwa-meth (Version:0.2.0) to compare the data after sequencing. According to the fetal-specific single-molecule methylation markers found in Example 2, use samtools (Version: 1.3.1) {Heng Li et al. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics} to extract all coverage DNA fragments of fetal-specific methylation markers. Use the epiread command of the biscuit toolkit (Version:0.2.0.20161222) to extract the single-molecule methylation pattern of the above DNA fragment. Through the analysis of the statistical software package MASS (Version: 7.3.51.1) in R language, fragments corresponding to the methylation markers of this model were obtained.
实验结果：在基因组(GRCh37版本)α-珠蛋白基因组区域上，寻找到的胎儿特异单分子甲基化模式，如图3中的A部分及表6所示。(图3中列出了原始测序结果，其中甲基化修饰以黑色表示，去甲基化修饰以白色表示，其它碱基以灰色表示，上方的分布图为每个碱基上平均的甲基化修饰情况，下方的DNA片段图为每条原始测序所得片段上甲基化模式；表6中列出了α-珠蛋白基因组区域内胎盘特异的单分子甲基化模式，第一列表示的是特定的单分子甲基化状态，其中，-1表示该位点发生去甲基化，1表示该位点发生甲基化修饰；第二列是单分子甲基化状态所在的具体的基因组位置信息，例如-1，-1/16，221645,221665表示的是16号染色体第221645个碱基和第221665个碱基同时发生去甲基化修饰，而这是胎盘特异的单分子甲基化模式。需注意的是，其中CpG位点包含正负两条链的单分子甲基化模式。)特别地，我们 发现，在第16号染色体第190000-230000碱基对位置之间，存在特异的胎儿单分子甲基化标志物，如图3中的B部分所示。这些单分子甲基化标志物，只在α-珠蛋白基因组区域为野生型或杂合携带SEA缺失基因型的胎盘中可以被观察到，在纯合SEA缺失基因型的胎盘和未孕女性外周血中，并不能被观察到。该实验雄辩地证明了，申请人发现的单分子甲基化标志物，只能来自于胎儿基因组，不可能来自于母亲基因组，是特异的α-珠蛋白基因组区域胎儿来源DNA片段标记物。Experimental results: The fetal-specific single-molecule methylation pattern found in the genome (GRCh37 version) α-globin genome region is shown in Part A of Table 3 and Table 6. (The original sequencing results are listed in Figure 3, where the methylation modification is represented in black, the demethylation modification is represented in white, and the other bases are represented in gray. The distribution diagram above is the average methyl group on each base The DNA fragment map below shows the methylation pattern on each original sequenced fragment; Table 6 lists the single-molecule methylation patterns specific to the placenta in the α-globin genomic region. It is a specific single-molecule methylation state, where -1 indicates that the site is demethylated, and 1 indicates that the site is methylated; the second column is the specific genome where the single-molecule methylation state is located. Position information, such as -1, -1/16, 221645, 221665 indicates that the 221645th base and the 221665th base of chromosome 16 are demethylated at the same time, which is a placental-specific single-molecule methyl group It should be noted that the CpG site contains a single molecule methylation pattern of plus and minus two chains.) In particular, we found that between the 190000-230000 base pair positions on chromosome 16 Specific fetal single-molecule methylation markers are shown in Part B of Figure 3. These single-molecule methylation markers can only be observed in the placenta where the α-globin genomic region is wild-type or heterozygous with SEA-deficient genotypes. It cannot be observed in the blood. The experiment eloquently proved that the single-molecule methylation markers discovered by the applicant can only come from the fetal genome, but not from the maternal genome, and are specific markers of DNA fragments derived from the fetus of the α-globin genome region.
表6胎儿特异单分子甲基化模式(其中，表6中的左栏是“特定的单分子甲基化状态”，右栏是“单分子甲基化状态所在的具体的基因组位置信息”)Table 6 Fetal-specific single-molecule methylation patterns (wherein the left column in Table 6 is "specific single-molecule methylation status" and the right column is "specific genomic location information where single-molecule methylation status is located")
Figure PCTCN2019123983-appb-000006
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实施例5：利用胎盘特异的甲基化模式，分析胎儿基因型已知的孕妇血Example 5: Analysis of blood of pregnant women with known fetal genotype using the methylation pattern specific to the placenta 浆DNAPlasma DNA
1.实验设计：从广州市妇女儿童健康中心产前诊断科招募受试者。本研究和人类临床样本的采集得到机构审查委员会的批准，并获得每位受试者的知情同意书。所收集样本为已做过羊水穿刺，并得知胎儿基因型( α-珠蛋白 基因缺失与否)的孕妇(11-14周孕龄)外周血样本(10mL，Streck 218962 10mL Cell-Free DNA BCT无创采血管)。 1. Experimental design: recruit subjects from the prenatal diagnosis department of Guangzhou Women and Children Health Center. The study and the collection of human clinical samples were approved by the institutional review board, and each subject’s informed consent was obtained. The samples were collected as amniocentesis done, and that the fetal genotype (globin gene deletion alpha] or not) pregnant women (gestational age 11-14 weeks) peripheral blood samples (10mL, Streck 218962 10mL Cell- Free DNA BCT Non-invasive blood collection tube).
2.WGBS甲基化测序：方法同实施例1中的相应部分。2. WGBS methylation sequencing: the method is the same as the corresponding part in Example 1.
3.单分子甲基化模式的统计3. Statistics of single molecule methylation pattern
测序后分析：使用bwa-meth(Version:0.2.0)对测序后数据进行比对。根据前述发现的α-珠蛋白基因组区域及对照区域上的胎儿特异单分子甲基化标志物，使用samtools(Version:1.3.1){Heng Li et al.(2009).The Sequence Alignment/Map format and SAMtools.Bioinformatics}提取比对到目标区域和对照区域的所有DNA片段。使用biscuit工具包(Version:0.2.0.20161222)的epiread命令，提取上述DNA片段的单分子甲基化模式。通过R语言的统计软件包MASS(Version：7.3.51.1)分析，获得符合胎儿特异甲基化模式标志物的片段。Post-sequencing analysis: use bwa-meth(Version:0.2.0) to compare the data after sequencing. According to the fetal-specific single-molecule methylation markers found on the α-globin genomic region and the control region, use samtools (Version: 1.3.1) {Heng Li Li et al. (2009). The Sequence Alignment/Map format and SAMtools.Bioinformatics} extract all DNA fragments that are compared to the target area and the control area. Use the epiread command of the biscuit toolkit (Version:0.2.0.20161222) to extract the single-molecule methylation pattern of the above DNA fragment. According to the analysis of the statistical software package MASS (Version: 7.3.51.1) in R language, fragments corresponding to the markers of fetal specific methylation pattern were obtained.
统计分析：根据上述计算的胎儿特异甲基化模式标志物计算结果，对α-珠蛋白基因组区域及对照区域，分别计算局部胎源比例，根据对照区域的局部胎源比例，对α-珠蛋白基因组区域的局部胎源比例作归一化处理。Statistical analysis: According to the calculation results of the fetal specific methylation pattern markers calculated above, the local fetal proportion is calculated for the α-globin genomic region and the control region, and the α-globin is calculated according to the local fetal proportion in the control region The proportion of local fetal origin in the genomic region is normalized.
实验结果：实验结果如图4所示。在68例孕妇血浆中，携带纯合SEA缺失基因型胎儿的孕妇，其归一化后的α-珠蛋白基因组区域局部胎源比例显著地低于其他孕妇。Experimental results: The experimental results are shown in Figure 4. In the plasma of 68 pregnant women, the pregnant women with homozygous SEA-deficient genotype fetuses had significantly lower localized fetal proportions in the normalized α-globin genomic region than other pregnant women.
实施例6：利用胎盘特异的甲基化模式，测试胎儿基因型未知的孕妇血Example 6: Using the specific methylation pattern of the placenta to test the blood of pregnant women with unknown fetal genotype 浆DNAPlasma DNA
1.实验设计：从广州市妇女儿童健康中心产前诊断科招募受试者。本研究和人类临床样本的采集得到机构审查委员会的批准，并获得每位受试者的知情同意书。在孕中期(11-14周孕龄)在超声引导下采集胎盘绒毛。在即将进行手术前采集母体外周血样本(10mL，Streck 218962 10mL Cell-Free DNA BCT无创采血管)。1. Experimental design: recruit subjects from the prenatal diagnosis department of Guangzhou Women and Children Health Center. The study and the collection of human clinical samples were approved by the institutional review board, and each subject’s informed consent was obtained. In the second trimester (11-14 weeks gestational age), placental villi were collected under ultrasound guidance. A maternal peripheral blood sample (10 mL, Streck 218962 10 mL Cell-Free DNA BCT non-invasive blood collection tube) was collected just before the operation.
2.WGBS甲基化测序：方法同实施例1中的相应部分。2. WGBS methylation sequencing: the method is the same as the corresponding part in Example 1.
3.绒毛基因型的判定：方法同实施例4中的相应部分。3. Determination of villi genotype: the method is the same as the corresponding part in Example 4.
4.利用 胎盘特异的甲基化模式预测胎儿基因型 4. The use of placenta-specific methylation patterns predict fetal genotype
测序后分析：方法同实施例5中的相应部分。Analysis after sequencing: The method is the same as the corresponding part in Example 5.
统计分析：根据计算结果，对α-珠蛋白基因组区域及对照区域，分别计算局部胎源比例，根据对照区域的局部胎源比例，对α-珠蛋白基因组区域的局部胎源比例作归一化处理。定义归一后的α-珠蛋白基因组区域局部胎源比例低于0.01的样本，为可能携带纯合SEA缺失基因型胎儿(即，重度α-地贫患儿)的孕妇。Statistical analysis: According to the calculation results, calculate the local fetal proportion for the α-globin genomic region and the control region, and normalize the local fetal proportion for the α-globin genomic region according to the local fetal proportion in the control region deal with. The normalized sample with a local fetal ratio of less than 0.01 in the α-globin genomic region is a pregnant woman who may carry a fetus with a homozygous SEA deletion genotype (ie, a child with severe α-thalassaemia).
结果：实验结果如表7和图5所示。在141例孕妇血浆中，经羊水穿刺检验揭盲，共正确鉴定出纯合SEA缺失胎儿50例，正确率99％(50/51)，正确鉴定出携带杂合SEA缺失基因型或完全野生型胎儿89例，正确率99％(89/90)。其敏感性为98.04％，特异性为98.89％。其中，未检出的一例重型地贫患儿，为胎盘嵌合个体。因此，本专利所述方法可于产前无创检测孕妇所怀胎儿是否患有重度α-地贫。Results: The experimental results are shown in Table 7 and Figure 5. In the plasma of 141 pregnant women, 50 patients with homozygous SEA-deleted fetuses were correctly identified by amniotic fluid puncture test, with a correct rate of 99% (50/51), and correctly identified the heterozygous SEA-deleted genotype or completely wild-type There were 89 fetuses with a correct rate of 99% (89/90). The sensitivity is 98.04% and the specificity is 98.89%. Among them, an undetected child with severe thalassemia was an individual with placenta fitting. Therefore, the method described in this patent can non-invasively detect whether the fetus is pregnant with severe α-thalassaemia before delivery.
表7孕妇怀孕胎儿检测结果Table 7 Test results of pregnant fetuses in pregnant women
Figure PCTCN2019123983-appb-000030
Figure PCTCN2019123983-appb-000030
Figure PCTCN2019123983-appb-000031
Figure PCTCN2019123983-appb-000031
Figure PCTCN2019123983-appb-000032
Figure PCTCN2019123983-appb-000032
Figure PCTCN2019123983-appb-000033
Figure PCTCN2019123983-appb-000033
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案的限定。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案予以穷举。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。The above examples of the present disclosure are merely examples for clearly illustrating the present disclosure, and are not intended to limit the embodiments of the present disclosure. For those of ordinary skill in the art, other different forms of changes or changes can be made based on the above description. There is no need to exhaustively implement all implementation options. Any modification, equivalent replacement and improvement made within the spirit and principle of the present disclosure shall be included in the protection scope of the claims of the present disclosure.

Claims (42)

  1. 一种检测胎儿特异性DNA甲基化模式的试剂在制备用于检测胎儿是否患有遗传疾病或患有遗传疾病的风险度的试剂或试剂盒中的用途，所述胎儿特异性DNA甲基化模式为DNA单分子甲基化状态；其中，所述DNA单分子甲基化状态的甲基化位点选自编码α-珠蛋白基因的区域；优选的，所述胎儿遗传疾病为α-地中海贫血。Use of a reagent for detecting a fetal-specific DNA methylation pattern in the preparation of a reagent or kit for detecting whether a fetus has a genetic disease or a risk of having a genetic disease, the fetal-specific DNA methylation The mode is a DNA single molecule methylation state; wherein, the methylation site of the DNA single molecule methylation state is selected from the region encoding the α-globin gene; preferably, the fetal genetic disease is α-Mediterranean anemia.
  2. 根据权利要求1所述的用途，其中，所述编码α-珠蛋白基因的区域选自人类第16号染色体190000-250000碱基对之间的区域；优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体如下位置中的任意一个碱基对：202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合。The use according to claim 1, wherein the region encoding the α-globin gene is selected from the region between 190000-250000 base pairs of human chromosome 16; preferably, the gene encoding the α-globin gene Is selected from any one of the base pairs of human chromosome 16 at the following positions: 202146, 202148, 202161, 202170, 202178, 202310, 202425, 205180, 205229, 205234, 205245, 209848, 209885, 209922, 209959, 209990 ,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763 ,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665 , 221709, 221740, 221748, 221767, 221800, 221810, 229406, 229454, 229484, 229499, 229527, 229535, or any combination of two or more of the above base pairs.
  3. 根据权利要求1所述的用途，其中，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合；优选的，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499或上述碱基对的组合。The use according to claim 1, wherein the region encoding the α-globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484, 229499, 229527, 229535 or the above base A combination of any two or more than two base pairs; preferably, the region encoding the α-globin gene contains at least any base selected from the following positions on human chromosome 16 Pair: 229484,229499 or a combination of the above base pairs.
  4. 根据权利要求1所述的用途，其中，所述检测胎儿特异性DNA甲基化模式的试剂选自DNA甲基化状态显示剂和/或检测来自孕妇的生物样本中胎儿特异性甲基化模式DNA的存在的试剂；优选的，所述检测所述来自孕妇的生物样本中胎儿特异性甲基化模式DNA的存在的试剂选自富集特征游离 DNA所需的试剂。The use according to claim 1, wherein the reagent for detecting fetal-specific DNA methylation patterns is selected from DNA methylation status indicators and/or for detecting fetal-specific methylation patterns in biological samples from pregnant women A reagent for the presence of DNA; preferably, the reagent for detecting the presence of fetal-specific methylation pattern DNA in the biological sample from a pregnant woman is selected from reagents required for enriching characteristic free DNA.
  5. 根据权利要求4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。The use according to claim 4, wherein the DNA methylation status display agent is selected from antibodies or binding proteins that recognize methylated DNA, bisulfite, enzymes with catalytic oxidation of DNA, with DNA desorption Amino-acting enzymes or methylation-sensitive enzymes, or a combination thereof; preferably, the methylation-sensitive enzyme is selected from methylation-sensitive restriction enzymes, more preferably, the methylation-sensitive enzyme The restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
  6. 根据权利要求4所述的用途，其中，所述富集特征游离DNA所需的试剂选自液相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。The use according to claim 4, wherein the reagents required for enriching characteristic free DNA are selected from reagents required for liquid phase hybridization probe capture method, reagents required for polymerase chain reaction amplification method, anchor The reagents required for the nucleic acid amplification method, the reagents used for sequencing-by-synthesis detection, or the reagents used for single-molecule sequencing, or a combination thereof.
  7. 根据权利要求4所述的试剂盒，其中，所述样本选自全血、外周血、血浆、血清、尿液、唾液、汗液，胎盘、胎盘绒毛、羊水或脐血，或其组合。The kit according to claim 4, wherein the sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid or cord blood, or a combination thereof.
  8. 根据权利要求1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。The use according to claim 1, wherein the test is a prenatal test; preferably, the prenatal test is a non-invasive prenatal test.
  9. 一种试剂盒，其用于检测来自孕妇的生物样本中胎儿特异性DNA甲基化模式的存在或不存在，所述试剂盒包含：A kit for detecting the presence or absence of fetal-specific DNA methylation patterns in biological samples from pregnant women, the kit comprising:
    (a)检测所述生物样本中胎儿特异性DNA甲基化模式的存在的试剂；(a) reagents for detecting the presence of fetal-specific DNA methylation patterns in the biological sample;
    可选的，所述试剂盒还包含：Optionally, the kit further includes:
    (b)DNA甲基化状态显示剂；(b) DNA methylation status indicator;
    其中，当(a)中的试剂不依赖于(b)中DNA甲基化状态显示剂时，试剂盒中不包含(b)；Where, when the reagent in (a) does not depend on the DNA methylation status indicator in (b), (b) is not included in the kit;
    优选的，所述胎儿特异性DNA甲基化模式为胎儿特异性DNA单分子甲基化状态。Preferably, the fetal-specific DNA methylation mode is a single-molecule methylation state of fetal-specific DNA.
  10. 根据权利要求9所述的试剂盒，其用于检测胎儿是否患有遗传疾病或患有遗传疾病的风险度；优选的，所述遗传疾病为α-地中海贫血。The kit according to claim 9, which is used to detect whether a fetus has a genetic disease or a risk of suffering from a genetic disease; preferably, the genetic disease is α-thalassemia.
  11. 根据权利要求9所述的试剂盒，其中，所述胎儿特异性DNA单分子甲基化状态的甲基化位点选自编码α-珠蛋白基因的区域；优选的，所述编码 α-珠蛋白基因的区域选自人类第16号染色体190000-250000碱基对之间的区域；更优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体如下位置中的任意一个碱基对：202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合。The kit according to claim 9, wherein the methylation site of the methylation status of the fetal-specific DNA single molecule is selected from the region encoding the α-globin gene; preferably, the encoding α-bead The region of the protein gene is selected from the region between 190000-250000 base pairs of human chromosome 16; more preferably, the region encoding the α-globin gene is selected from any base in the following position of human chromosome 16 Base pair: 202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247 ,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964 ,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454 , 229484, 229499, 229527, 229535 or any combination of two or more of the above base pairs.
  12. 根据权利要求9所述的试剂盒，其中，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合；优选的，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499或上述碱基对的组合。The kit according to claim 9, wherein the region encoding the α-globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484, 229499, 229527, 229535 or above Any two of the base pairs or a combination of more than two base pairs; preferably, the region encoding the α-globin gene contains at least any base selected from the following positions on human chromosome 16 Base pairs: 229484, 229499 or a combination of the above base pairs.
  13. 根据权利要求9所述的试剂盒，其中，所述样本选自全血、外周血、血浆、血清、尿液、唾液、汗液，胎盘、胎盘绒毛、羊水或脐血，或其组合。The kit according to claim 9, wherein the sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid, or cord blood, or a combination thereof.
  14. 根据权利要求9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。The kit according to claim 9, wherein the DNA methylation status display agent is selected from an antibody or a binding protein that recognizes methylated DNA, bisulfite, an enzyme that has catalytic oxidation of DNA, has 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 methylation-sensitive enzyme The restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
  15. 根据权利要求9所述的试剂盒，其中，所述富集特征游离DNA所需的试剂选自液相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。The kit according to claim 9, wherein the reagents required for enriching characteristic free DNA are selected from reagents required for liquid phase hybridization probe capture method, reagents required for polymerase chain reaction amplification method, Reagents required for the anchoring nucleic acid amplification method, reagents for sequencing-by-synthesis detection, reagents for single-molecule sequencing, or a combination thereof.
  16. 一种试剂盒，其用于检测胎儿是否患有遗传疾病或患有遗传疾病的风险度，所述试剂盒包含：A kit for detecting whether a fetus has a genetic disease or a risk of suffering from a genetic disease, the kit includes:
    (a)检测所述生物样本中胎儿特异性DNA甲基化模式的存在的试剂；(a) reagents for detecting the presence of fetal-specific DNA methylation patterns in the biological sample;
    可选的，所述试剂盒还包含：Optionally, the kit further includes:
    (b)DNA甲基化状态显示剂；(b) DNA methylation status indicator;
    其中，当(a)中的试剂不依赖于(b)中DNA甲基化状态显示剂时，试剂盒中不包含(b)；Where, when the reagent in (a) does not depend on the DNA methylation status indicator in (b), (b) is not included in the kit;
    优选的，所述胎儿特异性DNA甲基化模式为胎儿特异性DNA单分子甲基化状态。Preferably, the fetal-specific DNA methylation mode is a single-molecule methylation state of fetal-specific DNA.
  17. 根据权利要求16所述的试剂盒，其通过检测来自孕妇的生物样本中胎儿特异性DNA甲基化模式，进而检测胎儿是否患有遗传疾病或患有遗传疾病的风险度；优选的，所述遗传疾病为α-地中海贫血。The kit according to claim 16, which detects fetal specific DNA methylation patterns in biological samples from pregnant women, and thereby detects whether the fetus has a genetic disease or a risk of suffering from a genetic disease; preferably, the The genetic disease is α-thalassemia.
  18. 根据权利要求16所述的试剂盒，其中，所述单分子甲基化状态的甲基化位点选自编码α-珠蛋白基因的区域；优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体190000-250000碱基对之间的区域；更优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体如下位置中的任意一个碱基对：202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合。The kit according to claim 16, wherein the methylation site of the single-molecule methylation state is selected from the region encoding the α-globin gene; preferably, the region encoding the α-globin gene It is selected from the region between 190000-250000 base pairs of human chromosome 16; more preferably, the region encoding the α-globin gene is selected from any one of the base pairs of human chromosome 16 in the following position: 202146 ,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278 ,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104 ,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499 , 229527, 229535 or any combination of two or more of the above base pairs.
  19. 根据权利要求16所述的试剂盒，其中，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合；优选的，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499或上述碱基对的组合。The kit according to claim 16, wherein the region encoding the α-globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484, 229499, 229527, 229535 or above Any two of the base pairs or a combination of more than two base pairs; preferably, the region encoding the α-globin gene contains at least any base selected from the following positions on human chromosome 16 Base pairs: 229484, 229499 or a combination of the above base pairs.
  20. 根据权利要求16所述的试剂盒，其中，所述样本选自全血、外周血、血浆、血清、尿液、唾液、汗液，胎盘、胎盘绒毛、羊水或脐血，或其组合。The kit according to claim 16, wherein the sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid, or cord blood, or a combination thereof.
  21. 根据权利要求16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。The kit according to claim 16, wherein the DNA methylation status display agent is selected from an antibody or a binding protein that recognizes methylated DNA, bisulfite, an enzyme with catalytic oxidation of DNA, has 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 methylation-sensitive enzyme The restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
  22. 根据权利要求16所述的试剂盒，其中，所述富集特征游离DNA所需的试剂选自液相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。The kit according to claim 16, wherein the reagents required for enriching characteristic free DNA are selected from reagents required for the liquid phase hybridization probe capture method, reagents required for the polymerase chain reaction amplification method, Reagents required for the anchoring nucleic acid amplification method, reagents for sequencing-by-synthesis detection, reagents for single-molecule sequencing, or a combination thereof.
  23. 一种检测胎儿是否患有遗传疾病或患有遗传疾病的风险度的方法，所述方法包括下列步骤：A method for detecting whether a fetus has a genetic disease or a risk of genetic disease, the method includes the following steps:
    检测步骤：检测处理后的生物样本中总DNA片段的DNA甲基化模式；其中，所述的生物样本为怀孕女性血浆样本；Detection step: detecting the DNA methylation pattern of the total DNA fragments in the treated biological sample; wherein, the biological sample is a plasma sample of a pregnant female;
    区分步骤：区分所述检测步骤中得到的生物样本中总DNA片段的DNA甲基化的不同模式；Distinguishing step: distinguish different patterns of DNA methylation of the total DNA fragments in the biological sample obtained in the detection step;
    计算步骤：计算所述生物样本中携带特定DNA甲基化模式的DNA片段占该区域总DNA片段的比例，找到只存在于胎盘组织中而不存在于未孕女性血浆中的DNA甲基化模式，即胎儿特异的DNA甲基化模式；Calculation step: Calculate the proportion of DNA fragments carrying a specific DNA methylation pattern in the biological sample to the total DNA fragments in the area, and find the DNA methylation pattern that exists only in placental tissue but not in the plasma of infertile women , That is, fetal-specific DNA methylation pattern;
    统计步骤：根据找到的胎儿特异的DNA甲基化模式，统计孕妇血浆DNA样本在不同基因区域上胎儿来源的DNA片段占该区域总DNA片段的比例；Statistical steps: According to the fetal specific DNA methylation pattern found, the proportion of fetal DNA fragments in different gene regions of maternal plasma DNA samples in the total DNA fragments in this region is counted;
    比较步骤：比较孕妇血浆DNA样本比对到遗传疾病对应的基因区域内的胎儿来源的DNA片段占该区域总DNA片段数的比例与比对到对照基因区域内的胎儿来源的DNA片段占该区域总DNA片段数的比例；Comparison step: comparing the ratio of the DNA fragments of the pregnant women to the DNA fragments of the fetus in the gene region corresponding to the genetic disease to the total number of DNA fragments in the region and the DNA fragments of the fetus from the control gene region to the region The ratio of the total number of DNA fragments;
    可选的，所述方法还包括判断步骤：Optionally, the method further includes a judgment step:
    如果遗传疾病对应的基因区域内胎儿来源的DNA片段占总DNA片段数的比例与对照基因区域内胎儿来源的DNA片段占总DNA片段数的比例相比，存在不一致，则胎儿患有遗传疾病或具有高的患有遗传疾病的风险；If there is an inconsistency between the proportion of fetal-derived DNA fragments in the total DNA fragments in the gene region corresponding to the genetic disease and the proportion of fetal-derived DNA fragments in the control gene region, the fetus has a genetic disease or Have a high risk of suffering from genetic diseases;
    如果遗传疾病对应的基因区域内胎儿来源的DNA片段占总DNA片段数的比例与对照基因区域内胎儿来源的DNA片段占总DNA片段数的比例相比，不存在不一致，则胎儿未患有遗传疾病或不具有患有遗传疾病的风险或具有低的患有遗传疾病的风险。If the proportion of fetal-derived DNA fragments in the total DNA fragments in the gene region corresponding to the genetic disease is not inconsistent compared to the proportion of fetal-derived DNA fragments in the control gene region in the total DNA fragments, the fetus does not have genetic The disease may or may not have a risk of having a genetic disease or have a low risk of having a genetic disease.
  24. 根据权利要求23所述的方法，其中，所述检测步骤前，可包括处理步骤：使用DNA甲基化状态显示剂处理生物样本。The method according to claim 23, wherein before the detecting step, a processing step can be included: processing a biological sample using a DNA methylation status indicator.
  25. 根据权利要求23所述的方法，其中，所述计算步骤中，如果某个比对到遗传疾病对应的基因区域、且携带特定DNA甲基化模式的DNA片段只能在携带正常胎儿的孕妇的生物样本中被观察到，而不能从携带患有缺失型遗传疾病的胎儿的孕妇的生物样本中被观察到，则所述特定DNA甲基化模式的DNA片段只能为胎儿来源。The method according to claim 23, wherein, in the calculating step, if a certain DNA region corresponding to the genetic region corresponding to the genetic disease and carrying a specific DNA methylation pattern can only be used in pregnant women carrying normal fetuses Observed in biological samples, and not from biological samples of pregnant women carrying fetuses with deletion genetic diseases, the DNA fragments of the specific DNA methylation pattern can only be of fetal origin.
  26. 根据权利要求23所述的方法，其中，所述遗传疾病为α-地中海贫血。The method according to claim 23, wherein the genetic disease is α-thalassemia.
  27. 根据权利要求23所述的方法，其中，所述遗传疾病对应的基因区域选自编码α-珠蛋白基因的区域；优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体190000-250000碱基对之间的区域；更优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体如下位置中的任意一个碱基对：202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648, 210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合。The method according to claim 23, wherein the genetic region corresponding to the genetic disease is selected from the region encoding the α-globin gene; preferably, the region encoding the α-globin gene is selected from the human chromosome 16 A region between 190000-250000 base pairs; more preferably, the region encoding the α-globin gene is selected from any one of the base pairs in the following positions of human chromosome 16: 202146, 202148, 202161, 202170, 202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320, 210357,210362,210393,210398,210575,210617,210648, 210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185, 213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535 or the above bases A combination of any two or more than two base pairs in the base pair.
  28. 根据权利要求23所述的方法，其中，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合；优选的，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499或上述碱基对的组合。The method according to claim 23, wherein the region encoding the α-globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484, 229499, 229527, 229535 or the above base A combination of any two or more than two base pairs; preferably, the region encoding the α-globin gene contains at least any base selected from the following positions on human chromosome 16 Pair: 229484,229499 or a combination of the above base pairs.
  29. 根据权利要求23所述的方法，其中，所述样本选自全血、外周血、血浆、血清、尿液、唾液、汗液，胎盘、胎盘绒毛、羊水或脐血，或其组合。The method of claim 23, wherein the sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid, or cord blood, or a combination thereof.
  30. 根据权利要求23所述的方法，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。The method according to claim 23, wherein the DNA methylation status display agent is selected from an antibody or a binding protein that recognizes methylated DNA, bisulfite, an enzyme with DNA catalytic oxidation, and has a DNA desorption Amino-acting enzymes or methylation-sensitive enzymes, or a combination thereof; preferably, the methylation-sensitive enzyme is selected from methylation-sensitive restriction enzymes, more preferably, the methylation-sensitive enzyme The restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
  31. 根据权利要求23所述的方法，其中，所述富集特征游离DNA所需的试剂选自液相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。The method according to claim 23, wherein the reagents required for enriching characteristic free DNA are selected from reagents required for liquid phase hybridization probe capture method, reagents required for polymerase chain reaction amplification method, anchor The reagents required for the nucleic acid amplification method, the reagents for sequencing-by-synthesis detection, or the reagents for single-molecule sequencing, or a combination thereof.
  32. 一种检测胎儿是否患有遗传疾病或患有遗传疾病的风险度的检测系统，其中，所述检测系统包括如下模块：A detection system for detecting whether a fetus has a genetic disease or a risk of suffering from a genetic disease, wherein the detection system includes the following modules:
    检测模块：所述检测模块检测处理后的生物样本中总DNA片段的DNA甲基化模式；其中，所述的生物样本为怀孕女性血浆样本；Detection module: the detection module detects the DNA methylation pattern of the total DNA fragments in the processed biological sample; wherein, the biological sample is a plasma sample of a pregnant female;
    区分模块：所述区分模块区分所述检测步骤中得到的生物样本中总DNA 片段的DNA甲基化的不同模式；Differentiation module: the differentiation module distinguishes different patterns of DNA methylation of the total DNA fragments in the biological sample obtained in the detection step;
    计算模块：所述计算模块计算所述生物样本中携带特定DNA甲基化模式的DNA片段占该区域总DNA片段的比例，找到只存在于胎盘组织中而不存在于未孕女性血浆中的DNA甲基化模式，即胎儿特异的DNA甲基化模式；Calculation module: The calculation module calculates the proportion of DNA fragments carrying a specific DNA methylation pattern in the biological sample to the total DNA fragments in the region, and finds DNA that exists only in placental tissue but not in the plasma of infertile women Methylation mode, that is, fetal-specific DNA methylation mode;
    统计模块：所述统计模块根据找到的胎儿特异的DNA甲基化模式，统计孕妇血浆DNA样本在不同基因区域上胎儿来源的DNA片段占该区域总DNA片段的比例；Statistic module: The statistic module counts the proportion of fetal-derived DNA fragments of different maternal plasma DNA samples in different gene regions to the total DNA fragments in the region based on the found fetal-specific DNA methylation patterns;
    比较模块：所述比较模块比较孕妇血浆DNA样本比对到遗传疾病对应的基因区域内的胎儿来源的DNA片段占该区域总DNA片段数的比例与比对到对照基因区域内的胎儿来源的DNA片段占该区域总DNA片段数的比例；Comparison module: the comparison module compares the ratio of the DNA fragments of the fetus in the gene region corresponding to the genetic disease to the DNA fragments of the fetus in the genetic region corresponding to the genetic disease and the DNA from the fetus in the control gene region The proportion of fragments in the total number of DNA fragments in the region;
    可选的，所述系统还可以包括判断模块：Optionally, the system may further include a judgment module:
    所述判断模块进行如下判断：The judgment module makes the following judgments:
    如果遗传疾病对应的基因区域内胎儿来源的DNA片段占总DNA片段数的比例与对照基因区域内胎儿来源的DNA片段占总DNA片段数的比例相比，存在不一致，则胎儿患有遗传疾病或具有高的患有遗传疾病的风险；If there is an inconsistency between the proportion of fetal-derived DNA fragments in the total DNA fragments in the gene region corresponding to the genetic disease and the proportion of fetal-derived DNA fragments in the control gene region, the fetus has a genetic disease or Have a high risk of suffering from genetic diseases;
    如果遗传疾病对应的基因区域内胎儿来源的DNA片段占总DNA片段数的比例与对照基因区域内胎儿来源的DNA片段占总DNA片段数的比例相比，不存在不一致，则胎儿未患有遗传疾病或不具有患有遗传疾病的风险或具有低的患有遗传疾病的风险。If the proportion of fetal-derived DNA fragments in the total DNA fragments in the gene region corresponding to the genetic disease is not inconsistent compared to the proportion of fetal-derived DNA fragments in the control gene region in the total DNA fragments, the fetus does not have genetic The disease may or may not have a risk of having a genetic disease or have a low risk of having a genetic disease.
  33. 根据权利要求32所述的检测系统，其中，所述检测系统还包括处理模块：所述处理模块使用DNA甲基化状态显示剂处理生物样本。The detection system according to claim 32, wherein the detection system further comprises a processing module: the processing module processes a biological sample using a DNA methylation status indicator.
  34. 根据权利要求32所述的系统，其中，所述计算模块中，如果某个比对到遗传疾病对应的基因区域、且携带特定DNA甲基化模式的DNA片段只能在携带正常胎儿的孕妇的生物样本中被观察到，而不能从携带患有缺失型遗传疾病的胎儿的孕妇的生物样本中被观察到，则所述特定DNA甲基化模式的DNA片段只能为胎儿来源。The system according to claim 32, wherein in the calculation module, if a certain DNA region corresponding to the genetic region corresponding to the genetic disease and carrying a specific DNA methylation pattern can only be used in pregnant women carrying normal fetuses Observed in biological samples, and not from biological samples of pregnant women carrying fetuses with deletion genetic diseases, the DNA fragments of the specific DNA methylation pattern can only be of fetal origin.
  35. 根据权利要求32所述的系统，其中，所述遗传疾病为α-地中海贫血。The system according to claim 32, wherein the genetic disease is α-thalassemia.
  36. 根据权利要求32所述的系统，其中，所述遗传疾病对应的基因区域选自编码α-珠蛋白基因的区域；优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体190000-250000碱基对之间的区域；更优选的，所述编码α-珠蛋白基因的区域选自人类第16号染色体如下位置中的任意一个碱基对：202146,202148,202161,202170,202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320,210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185,213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合。The system according to claim 32, wherein the genetic region corresponding to the genetic disease is selected from the region encoding the α-globin gene; preferably, the region encoding the α-globin gene is selected from the human chromosome 16 A region between 190000-250000 base pairs; more preferably, the region encoding the α-globin gene is selected from any one of the base pairs in the following positions of human chromosome 16: 202146, 202148, 202161, 202170, 202178,202310,202425,205180,205229,205234,205245,209848,209885,209922,209959,209990,209995,210027,210032,210063,210068,210210,210215,210247,210252,210278,210284,210289,210320, 210357,210362,210393,210398,210575,210617,210648,210653,210690,210721,210726,210763,210800,210821,210832,210834,210837,210868,210873,210964,210974,213104,213116,213178,213185, 213194,213200,216200,216231,216243,216483,216503,216505,221552,221560,221645,221665,221709,221740,221748,221767,221800,221810,229406,229454,229484,229499,229527,229535 or the above bases A combination of any two or more than two base pairs in the base pair.
  37. 根据权利要求32所述的系统，其中，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499,229527,229535或上述碱基对中的任意两者或多于任意两者的碱基对的组合；优选的，所述编码α-珠蛋白基因的区域至少包含选自人类第16号染色体如下位置中的任意一个碱基对：229484,229499或上述碱基对的组合。The system according to claim 32, wherein the region encoding the α-globin gene comprises at least one base pair selected from the following positions of human chromosome 16: 229484, 229499, 229527, 229535 or the above base A combination of any two or more than two base pairs; preferably, the region encoding the α-globin gene contains at least any base selected from the following positions on human chromosome 16 Pair: 229484,229499 or a combination of the above base pairs.
  38. 根据权利要求32所述的系统，其中，所述样本选自全血、外周血、血浆、血清、尿液、唾液、汗液，胎盘、胎盘绒毛、羊水或脐血，或其组合。The system of claim 32, wherein the sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid, or cord blood, or a combination thereof.
  39. 根据权利要求32所述的系统，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。The system according to claim 32, wherein the DNA methylation status display agent is selected from an antibody or a binding protein that recognizes methylated DNA, bisulfite, an enzyme that has catalytic oxidation of DNA, and has DNA removal Amino-acting enzymes or methylation-sensitive enzymes, or a combination thereof; preferably, the methylation-sensitive enzyme is selected from methylation-sensitive restriction enzymes, more preferably, the methylation-sensitive enzyme The restriction enzyme is selected from HpaII or BstUI, or a combination thereof.
  40. 根据权利要求32所述的系统，其中，所述富集特征游离DNA所需的 试剂选自液相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。The system according to claim 32, wherein the reagents required for enriching characteristic free DNA are selected from reagents required for liquid phase hybridization probe capture method, reagents required for polymerase chain reaction amplification method, anchor The reagents required for the nucleic acid amplification method, the reagents for sequencing-by-synthesis detection, or the reagents for single-molecule sequencing, or a combination thereof.
  41. 一种用于检测胎儿是否患有遗传疾病或患有遗传疾病的风险度的检测设备，包括：A detection device for detecting whether a fetus has a genetic disease or a risk of genetic disease, including:
    处理器；processor;
    用于存储处理器和执行指令的存储器；Memory for storing processors and executing instructions;
    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23-31任一项所述的方法。Wherein, the processor is configured to implement the method of any one of claims 23-31 when executing the processor-executable instructions.
  42. 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23-31任一项所述的方法。A non-volatile computer-readable storage medium having computer program instructions stored thereon, when the computer program instructions are executed by a processor, implement the method of any one of claims 23-31.
PCT/CN2019/123983 2018-12-13 2019-12-09 Method for non-invasive prenatal testing of fetus for genetic disease WO2020119626A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024114253A1 (en) * 2022-11-28 2024-06-06 广东菲鹏生物有限公司 Kit for determining whether individual to which sample to be tested belongs has genetic disease

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1352956A1 (en) * 1993-11-30 2003-10-15 McGILL UNIVERSITY Inhibition of DNA methyltransferase
CN103421903A (en) * 2013-08-14 2013-12-04 邯郸市康业生物科技有限公司 Alpha-thalassemia screening kit and application thereof in prenatal screening
CN108642160A (en) * 2018-05-16 2018-10-12 广州市达瑞生物技术股份有限公司 Detect the method and kit of fetus thalassemia Disease-causing gene

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100557033C (en) * 2007-04-25 2009-11-04 亚能生物技术（深圳）有限公司 Diagnosing alpha-thalassemic nucleic acid film bar and test kit

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1352956A1 (en) * 1993-11-30 2003-10-15 McGILL UNIVERSITY Inhibition of DNA methyltransferase
CN103421903A (en) * 2013-08-14 2013-12-04 邯郸市康业生物科技有限公司 Alpha-thalassemia screening kit and application thereof in prenatal screening
CN108642160A (en) * 2018-05-16 2018-10-12 广州市达瑞生物技术股份有限公司 Detect the method and kit of fetus thalassemia Disease-causing gene

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NICHOLAS, P.A. ET AL.: "Acquired A-Thalassemia in Preleukemia is Due to Decreased Expression of All Four A-Globin Genes", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 80, 31 October 1983 (1983-10-31), XP055713543, DOI: 20200224160652 *
刘莹等 (LIU, YING ET AL.): "重型β地中海贫血γ珠蛋白基因启动子区甲基化状态 (Methylation Status of γ-Globin Gene Promoter in β-Thalassemia Major)", 中国实验血液学杂志 (JOURNAL OF EXPERIMENTAL HEMATOLOGY), vol. 20, no. 3, 31 December 2012 (2012-12-31), DOI: 20200224164053 *
杜萌等 (DU, MENG ET AL.): "地中海贫血致病机制及基因治疗进展 (Thalassemia Pathogenesis and Gene Therapy Progress)", 医学动物防制 (JOURNAL OF MEDICAL PEST CONTROL), vol. 33, no. 1, 31 January 2017 (2017-01-31), DOI: 20200224160327 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024114253A1 (en) * 2022-11-28 2024-06-06 广东菲鹏生物有限公司 Kit for determining whether individual to which sample to be tested belongs has genetic disease

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