CN111321210B

CN111321210B - Method for non-invasive prenatal detection of whether fetus suffers from genetic disease

Info

Publication number: CN111321210B
Application number: CN201811528552.4A
Authority: CN
Inventors: 张翼; 刘九思; 常璐媛
Original assignee: Guiyang Youle Fusheng Medical Laboratory Co ltd
Current assignee: Ankang Youle Fusheng Technology Co.,Ltd.
Priority date: 2018-12-13
Filing date: 2018-12-13
Publication date: 2021-09-14
Anticipated expiration: 2038-12-13
Also published as: WO2020119626A1; CN111321210A

Abstract

The present disclosure relates to the 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 is at risk of having a genetic disease. The present disclosure also relates to a method for non-invasive prenatal detection of whether a fetus is suffering from a genetic disease, a detection kit for the method, a related detection method and detection system, a readable storage medium corresponding to the detection system, and a detection device. The detection kit, the related detection method, the detection system, the readable storage medium corresponding to the detection system and the detection equipment provided by the disclosure can be used for more accurately, more efficiently and more conveniently diagnosing the fetal genetic diseases, particularly the alpha-thalassemia in a non-invasive manner.

Description

Method for non-invasive prenatal detection of whether fetus suffers from genetic disease

Technical Field

The present disclosure relates to methylation modifications on a range of human genomic sites and combinations thereof that can be used for non-invasive prenatal diagnosis and screening. More particularly, the disclosure relates to a series of combinations of human genome site methylation modifications with placental tissue specificity, which can be used for non-invasive prenatal diagnosis and screening whether a fetus has a genetic disease or the risk of having a genetic disease, such as non-invasive diagnosis and screening of alpha thalassemia. The disclosure also relates to an experimental method for non-invasive prenatal diagnosis and screening of alpha thalassemia based on the series of genome locus methylation modifications, and a related high-throughput sequencing kit.

Background

Alpha-thalassemia (alpha-thalassemia, abbreviated as alpha-thalassemia) is hereditary anemia caused by a reduction or inhibition of hemoglobin alpha peptide chain synthesis due to alpha-globin gene mutation or deletion. Alpha-thalassemia occurs mostly in tropical and subtropical regions, such as southeast asia, indian subcontinent and south china. Worldwide, α -thalassemia is one of the most common monogenic genetic diseases, with a 5% carrier of α -thalassemia genes. In mainland China, the carrying rate of the alpha-thalassemia gene is 7.88 percent, and the carrying rate of the alpha-thalassemia gene in Guangxi province is as high as 14.13 percent.

The clinical manifestations of α -thalassemia are directly related to the number of α -globin genes, and when α -globin genes are completely deleted (-/-, severe α -thalassemia), patients develop disease during fetal period, and the symptoms of severe anemia with systemic edema (HbBarts' hydropsfetalis, babysia edema syndrome) are shown. The disease is fatal to the fetus and can cause serious and even life-threatening complications to the mother, and if adequate medical care is not available, the mortality rate of pregnant women can approach 50%. Furthermore, since the endemic area of α -thalassemia coincides with the malaria endemic area, the genetic disease poses a heavy public health burden especially in less developed countries and regions.

At present, the standard prenatal diagnosis method of alpha-thalassemia is to perform genotyping screening on high-risk couples and further perform prenatal diagnosis on placenta or fetal cells by methods such as villus biopsy and amniocentesis (Benz EJ Jr.2011.New born screening for a-third embedding with diagnosis. NEngl JMed364: 770-. However, these conventional methods are invasive and may cause damage to the fetus and pregnant woman, and even risk miscarriage of the fetus, while also increasing the psychological burden on the pregnant woman. There are studies showing surgical abortion risks of 0.22% and 0.11% for villus biopsy and amniocentesis, respectively (Akolekar et al, Ultrasound ObstetGynecol 2015,45(1): 16-26).

The discovery of Fetal cell-free DNA (cffDNA) in the peripheral blood of pregnant women makes possible Non-invasive Prenatal Testing (NIPT) of Fetal genetic status with cffDNA as a marker. Compared with the traditional invasive method, the NIPT has the advantages of safety, no complication, early diagnosis and the like, and has been successfully applied to fetal sex determination, fetal RhD genotype determination, and fetal chromosome aneuploidy screening (such as Down syndrome, Edward syndrome and Paotto syndrome). There have also been some scientific findings using cffDNA for non-invasive prenatal diagnosis of monogenic genetic diseases such as beta-thalassemia and Wilson's disease (Chiu et al, Lancet 360: 998-.

However, until now, noninvasive diagnosis of α -thalassemia has presented significant challenges. Severe α -thalassemia (-/-) is caused by long fragment deletion types of α -globin genes (e.g., -SEA subtypes), while Cell-free DNA (cfDNA, also known as free DNA) is in a highly fragmented state (Lo, y.m.d.et al (2010) sci.trans.med.2, 61ra91), and thus, conventional α -thalassemia detection methods, such as Gap polymerase chain reaction (Gap-PCR), multiple ligation probe amplification technology (pa MLPA), etc., are not suitable for noninvasive detection of α -thalassemia genes.

Studies have shown that fetuses that partially inherit the paternal wild-type alpha-globin allele can be excluded by detecting alpha-globin gene-associated, paternally-specific microsatellite sequences or Single Nucleotide Polymorphism Sites (SNPs) in maternal free DNA, but that at least 50% of fetuses are not genotyped (Ho et al, (2010) prenatdiagn.30(1): 65-73.; TZ Yan et al, (2011) PLoS one.6(9): e 24779). The assessment of the relative content of alpha-globin gene in maternal free DNA using real-time PCR or high throughput targeted sequencing techniques based on allele dose can be used to determine the genotype of fetal alpha-globin gene, but the accuracy of such methods depends on the fetal free DNA concentration (Long et al, (2009) zhonghua xue Ye xue zazhi.30(3):175-8 Ge et al, (2013) PLoS one.2013 Jun 28; 8(6): e 67464.). In addition to the above studies, recent studies have shown that the genotype of the fetal α -globin gene can be identified by analyzing the Relative haplotype dose (Relative haplotype dosage, RHDO) of the α -globin gene in maternal free DNA using SNP information of the haplotype region associated with the α -globin gene on the parental chromosome (W Wang et al, (2017) Genet Test Mol biomarkers.2017 Jul; 21(7): 433-439.). However, although the haploid typing technology has advanced sufficiently, the noninvasive diagnosis method based on the relative haplotype dosage needs to construct haploid models of both parents in advance, and the detection cost is high, the period is long, and the large-scale application is difficult.

That is, whether based on allelic balance or relative haplotype dose detection methods, they are essentially indirect methods of detecting fetal free DNA, and thus, maternal free DNA will generate a large background noise. Fetal DNA is not only present in low amounts in maternal plasma, but also co-exists with large amounts of maternal DNA (Lo YMD et al, Am J Hum Genet 1998; 62: 768-75; Lun, F.M et al, Clin. chem.2008,54, 1664-. Therefore, the maternal DNA background interferes more with the detection results. Although there are cases where noninvasive prenatal gene detection of fetal α -thalassemia was successfully performed, fetal free DNA concentration in the samples was high, with a median of 23.41%, far exceeding the mean level (Ge et al, PLoS one.2013; 8(6): e 67464). Therefore, in order to avoid maternal DNA interference and thereby more efficiently and accurately assess the genotype of the fetal α -globin gene, it is desirable to identify independent fetal DNA markers that are specific and independent of restriction factors such as fetal genomic polymorphic site or fetal gender.

Therefore, in order to avoid maternal DNA interference and to further more accurately, easily, economically and efficiently assess the genotype of the fetal α -globin gene and to perform non-invasive prenatal diagnosis of α -thalassemia, it is desirable to identify specific, independent fetal DNA markers that are independent of restriction factors such as fetal genomic polymorphic sites or fetal gender.

Disclosure of Invention

Problems to be solved by the invention

The present disclosure provides fetal DNA markers, methods of detection and kits useful as noninvasive prenatal genetic diagnosis of fetal alpha-thalassemia. Compared with the prior art, the technical scheme involved in the method can be used for carrying out noninvasive prenatal gene diagnosis on the fetal alpha-thalassemia more safely, efficiently, accurately and economically.

Means for solving the problems

The present disclosure includes, but is not limited to, the following technical solutions.

1. Use of a reagent for detecting a fetal-specific DNA methylation pattern that is a DNA single molecule methylation state in the manufacture of a reagent or kit for detecting whether a fetus has a genetic disease or is at risk of having a genetic disease; wherein the methylation site of the DNA unimolecular methylation state is selected from the region encoding the alpha-globin gene; preferably, the fetal genetic disease is α -thalassemia.

2. The use according to 1, wherein the region encoding the α -globin gene is selected from the region between 190000 and 250000 base pairs of human chromosome 16; preferably, the region encoding the α -globin gene is selected from any one of the base pairs of 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 any two or more of the base pairs described above.

3. The use according to any one of claims 1-2, wherein the region encoding the α -globin gene comprises at least one base pair selected from any one of the following positions of human chromosome 16: 229484,229499,229527,229535 or any two or more of the base pairs described above; preferably, the region encoding the α -globin gene comprises at least one base pair selected from any one of the following positions of human chromosome 16: 229484,229499 or a combination of the above base pairs.

4. The use according to any one of claims 1-3, wherein the agent for detecting a fetal-specific DNA methylation pattern is selected from a DNA methylation status indicator and/or an agent for detecting the presence of fetal-specific methylation pattern DNA in a biological sample from a pregnant woman; preferably, the reagents for detecting the presence of fetal-specific methylation pattern DNA in the biological sample from the pregnant woman are selected from the reagents required for enriching for characteristic free DNA.

5. The use according to any one of claims 1 to 4, wherein the DNA methylation status indicator is selected from an antibody or binding protein that recognizes methylated DNA, a bisulfite, an enzyme having DNA catalytic oxidation, an enzyme having DNA deamination or a methylation sensitive enzyme, 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.

6. The use according to any one of claims 1 to 5, wherein the reagents required for enriching for characteristic free DNA are selected from reagents required for a solution phase hybridization probe capture method, reagents required for a polymerase chain reaction amplification method, reagents required for an anchored nucleic acid amplification method, reagents for sequencing-by-synthesis detection, or reagents for single molecule sequencing, or a combination thereof.

7. The use of any one of claims 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 a combination thereof.

8. The use according to any one of claims 1 to 7, wherein the test is a prenatal test; preferably, the prenatal testing is non-invasive prenatal testing.

9. A kit for detecting the presence or absence of a fetal-specific DNA methylation pattern in a biological sample from a pregnant woman, the kit comprising:

(a) (ii) reagents for detecting the presence of a fetal-specific DNA methylation pattern in the biological sample;

optionally, the kit further comprises:

(b) a DNA methylation state indicator;

wherein (b) is not contained in the kit when the reagent in (a) is independent of the DNA methylation state indicator in (b);

preferably, the fetal-specific DNA methylation pattern is a fetal-specific DNA single-molecule methylation state.

10. The kit according to 9 for detecting whether a fetus has or is at risk of having a genetic disease; preferably, the genetic disease is α -thalassemia.

11. The kit of any one of claims 9-10, wherein the methylation site of the fetal-specific DNA single molecule methylation state is selected from the group consisting of a region encoding an alpha-globin gene; preferably, the region encoding the α -globin gene is selected from the region between the base pairs 190000-250000 of human chromosome 16; more preferably, the region encoding the α -globin gene is selected from any one of the base pairs of 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 any two or more of the base pairs described above.

12. The kit according to any one of claims 9 to 11, wherein the region encoding α -globin gene comprises at least any one base pair selected from the following positions of human chromosome 16: 229484,229499,229527,229535 or any two or more of the base pairs described above; preferably, the region encoding the α -globin gene comprises at least one base pair selected from any one of the following positions of human chromosome 16: 229484,229499 or a combination of the above base pairs.

13. The kit of any one of claims 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 a combination thereof.

14. The kit according to any one of claims 9 to 13, wherein the DNA methylation status indicator is selected from an antibody or binding protein that recognizes methylated DNA, a bisulfite, an enzyme having DNA catalytic oxidation, an enzyme having DNA deamination or a methylation sensitive enzyme, 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. The kit according to any one of claims 9 to 14, wherein the reagents required for enriching for characteristic free DNA are selected from reagents required for a solution phase hybridization probe capture method, reagents required for a polymerase chain reaction amplification method, reagents required for an anchored nucleic acid amplification method, reagents for sequencing-by-synthesis detection, or reagents for single molecule sequencing, or a combination thereof.

16. A kit for detecting whether a fetus has or is at risk of having a genetic disease, the kit comprising:

optionally, the kit further comprises:

(b) a DNA methylation state indicator;

17. The kit of 16, for detecting whether a fetus has a genetic disease or is at risk of having a genetic disease by detecting a fetal-specific DNA methylation pattern in a biological sample from a pregnant woman; preferably, the genetic disease is α -thalassemia.

18. The kit of any one of claims 16-17, wherein the methylation site of the single molecule methylation state is selected from the group consisting of a region encoding an alpha-globin gene; preferably, the region encoding the α -globin gene is selected from the region between the base pairs 190000-250000 of human chromosome 16; more preferably, the region encoding the α -globin gene is selected from any one of the base pairs of 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 any two or more of the base pairs described above.

19. The kit according to any one of claims 16 to 18, wherein the region encoding α -globin gene comprises at least any one base pair selected from the following positions of human chromosome 16: 229484,229499,229527,229535 or any two or more of the base pairs described above; preferably, the region encoding the α -globin gene comprises at least one base pair selected from any one of the following positions of human chromosome 16: 229484,229499 or a combination of the above base pairs.

20. The kit of any one of claims 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 a combination thereof.

21. The kit according to any one of claims 16-20, wherein the DNA methylation status indicator is selected from an antibody or binding protein that recognizes methylated DNA, a bisulfite, an enzyme having DNA catalytic oxidation, an enzyme having DNA deamination or a methylation sensitive enzyme, 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. The kit according to any one of claims 16 to 21, wherein the reagents required for enriching for characteristic free DNA are selected from reagents required for a solution phase hybridization probe capture method, reagents required for a polymerase chain reaction amplification method, reagents required for an anchored nucleic acid amplification method, reagents for sequencing-by-synthesis detection, or reagents for single molecule sequencing, or a combination thereof.

23. A method of detecting whether a fetus has a genetic disease or is at risk for having a genetic disease, the method comprising the steps of:

a 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;

a distinguishing step: differentiating the different patterns of DNA methylation of total DNA fragments in the biological sample obtained in the detecting step;

a calculation step: calculating the proportion of DNA fragments carrying a specific DNA methylation pattern in the biological sample to the total DNA fragments in the region, and finding out the DNA methylation pattern only existing in the placenta tissue and not existing in the plasma of the non-pregnant female, namely the DNA methylation pattern specific to the fetus;

a statistical step: according to the found fetal specific DNA methylation pattern, counting the proportion of fetal-derived DNA fragments of the pregnant woman plasma DNA sample in different gene regions in the total DNA fragments of the region;

a comparison step: comparing the proportion of the pregnant woman plasma DNA sample to the total DNA fragment number of the region of the fetal DNA fragment in the gene region corresponding to the genetic disease and the proportion of the fetal DNA fragment to the total DNA fragment number of the region in the gene region corresponding to the control gene region;

optionally, the method further includes a determining step:

if the proportion of the DNA fragments of fetal origin in the gene region corresponding to the genetic disease to the total number of DNA fragments is inconsistent with the proportion of the DNA fragments of fetal origin in the control gene region to the total number of DNA fragments, the fetus suffers from the genetic disease or has a high risk of suffering from the genetic disease;

if there is no inconsistency in the ratio of fetal-derived DNA fragments to the total number of DNA fragments in the gene region corresponding to the genetic disease as compared to the ratio of fetal-derived DNA fragments to the total number of DNA fragments in the control gene region, the fetus does not have the genetic disease or does not have the risk of having the genetic disease or has a low risk of having the genetic disease.

24. The method according to claim 23, wherein the detecting step may comprise, before the detecting step, a processing step of: the biological sample is treated with a DNA methylation state indicator.

25. The method according to any one of claims 23 to 24, wherein in the calculating step, the DNA fragments of the specific DNA methylation pattern are of fetal origin only if a certain alignment leads to a genetic region corresponding to a genetic disease and the DNA fragments carrying the specific DNA methylation pattern are only observable in a biological sample of a pregnant woman carrying a normal fetus and are not observable in a biological sample of a pregnant woman carrying a fetus with a deletion-type genetic disease.

26. The method of any one of claims 23-25, wherein the genetic disease is α -thalassemia.

27. The method according to any one of claims 23 to 26, wherein the genetic disease corresponds to a gene region selected from the group consisting of a region encoding the α -globin gene; preferably, the region encoding the α -globin gene is selected from the region between the base pairs 190000-250000 of human chromosome 16; more preferably, the region encoding the α -globin gene is selected from any one of the base pairs of 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 any two or more of the base pairs described above.

28. The method according to any one of claims 23 to 27, wherein the region encoding α -globin gene comprises at least one base pair selected from any one of the following positions of human chromosome 16: 229484,229499,229527,229535 or any two or more of the base pairs described above; preferably, the region encoding the α -globin gene comprises at least one base pair selected from any one of the following positions of human chromosome 16: 229484,229499 or a combination of the above base pairs.

29. The method of any one of claims 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 a combination thereof.

30. The method of any one of claims 23-29, wherein the DNA methylation status indicator is selected from an antibody or binding protein that recognizes methylated DNA, a bisulfite, an enzyme having DNA catalytic oxidation, an enzyme having DNA deamination, or a methylation sensitive enzyme, 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.

31. The method of any one of claims 23-30, wherein the reagents required for enriching for characteristic free DNA are selected from reagents required for a solution phase hybridization probe capture method, reagents required for a polymerase chain reaction amplification method, reagents required for an anchored nucleic acid amplification method, reagents for sequencing-by-synthesis detection, or reagents for single molecule sequencing, or a combination thereof.

32. A detection system for detecting whether a fetus has a genetic disease or is at risk of having a genetic disease, wherein the detection system comprises the following modules:

a detection module: the detection module detects the DNA methylation mode of the total DNA fragments in the processed biological sample; wherein the biological sample is a plasma sample of a pregnant female;

a distinguishing module: the distinguishing module distinguishes different modes of DNA methylation of total DNA fragments in the biological sample obtained in the detecting step;

a calculation module: the calculation module calculates the proportion of DNA fragments carrying specific DNA methylation patterns in the biological sample to the total DNA fragments in the region, and finds out the DNA methylation patterns only existing in the placenta tissues and not existing in the plasma of the non-pregnant women, namely the DNA methylation patterns specific to fetuses;

a statistic module: the statistical module is used for counting the proportion of fetal-derived DNA fragments of the maternal plasma DNA sample in different gene regions in the total DNA fragments of the region according to the found fetal-specific DNA methylation mode;

a comparison module: the comparison module compares the proportion of the pregnant woman plasma DNA sample to the total DNA fragment number of the region of the DNA fragment of the fetal source in the gene region corresponding to the genetic disease and the proportion of the DNA fragment of the fetal source in the control gene region to the total DNA fragment number of the region;

optionally, the system may further include a determining module:

the judgment module judges as follows:

33. The detection system of claim 32, wherein the detection system further comprises a processing module: the processing module processes the biological sample using a DNA methylation state indicator.

34. The detection system according to any one of claims 32-33, wherein the calculation module is configured to determine that the DNA fragments carrying the specific DNA methylation pattern are of fetal origin if a certain alignment identifies a genetic region corresponding to a genetic disease and the DNA fragments carrying the specific DNA methylation pattern are only observable in a biological sample of a pregnant woman carrying a normal fetus and not observable in a biological sample of a pregnant woman carrying a fetus with a deletion-type genetic disease.

35. The test system according to any one of claims 32-34, wherein the genetic disease is α -thalassemia.

36. A test system according to any one of claims 32-35, wherein the genetic disease-corresponding gene region is selected from the group consisting of a region encoding an alpha-globin gene; preferably, the region encoding the α -globin gene is selected from the region between the base pairs 190000-250000 of human chromosome 16; more preferably, the region encoding the α -globin gene is selected from any one of the base pairs of 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 any two or more of the base pairs described above.

37. The test system according to any one of claims 32 to 36, wherein the region encoding α -globin gene comprises at least one base pair selected from any one of the following positions of human chromosome 16: 229484,229499,229527,229535 or any two or more of the base pairs described above; preferably, the region encoding the α -globin gene comprises at least one base pair selected from any one of the following positions of human chromosome 16: 229484,229499 or a combination of the above base pairs.

38. A test system according to any one of claims 32-37, wherein the sample is selected from whole blood, peripheral blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid or umbilical cord blood, or a combination thereof.

39. A test system according to any one of claims 32-38, wherein the DNA methylation status indicator is selected from an antibody or binding protein that recognizes methylated DNA, a bisulphite, an enzyme having DNA catalytic oxidation, an enzyme having DNA deamination or a methylation sensitive enzyme, 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. The detection system according to any one of claims 32-39, wherein the reagents required for enriching for characteristic free DNA are selected from reagents required for a solution phase hybridization probe capture method, reagents required for a polymerase chain reaction amplification method, reagents required for an anchored nucleic acid amplification method, reagents for sequencing-by-synthesis detection, or reagents for single molecule sequencing, or a combination thereof.

41. A detection apparatus for detecting whether a fetus has a genetic disease or is at risk of having a genetic disease, comprising:

a processor;

a memory for storing a processor and executing instructions;

wherein the processor is configured to implement the method of any of the preceding 23-31 when executing the processor-executable instructions.

42. A non-transitory computer readable storage medium having stored thereon computer program instructions which, when executed by a processor, implement the method of any of the preceding 23-31.

In one aspect of the present disclosure, there is provided a method of detecting whether a fetus has a genetic disease or is at risk of having a genetic disease, the method comprising the steps of: detecting the DNA methylation pattern of the total DNA fragments in the treated biological sample; differentiating the different patterns of DNA methylation of total DNA fragments in the biological sample obtained in the detecting step; calculating the proportion of DNA fragments carrying a specific DNA methylation pattern in the biological sample to the total DNA fragments in the region, and finding out the DNA methylation pattern only existing in the placenta tissue and not existing in the plasma of the non-pregnant female, namely the DNA methylation pattern specific to the fetus; according to the found fetal specific DNA methylation pattern, counting the proportion of fetal-derived DNA fragments of the pregnant woman plasma DNA sample in different gene regions in the total DNA fragments of the region; comparing the proportion of the pregnant woman plasma DNA sample to the total DNA fragment number of the region of the fetal DNA fragment in the gene region corresponding to the genetic disease with the proportion of the fetal DNA fragment in the control gene region. In a preferred embodiment, the fetus is tested for genetic disease or for risk of genetic disease by determining the difference in the aforementioned ratio.

In another aspect of the disclosure, a method of detecting whether a fetus has or is at risk of having alpha-thalassemia is provided. The method comprises the following steps: detecting the methylation pattern of DNA in the biological sample; differentiating the different patterns of DNA methylation of total DNA fragments in the biological sample obtained in the detecting step; calculating the methylation pattern of the placenta tissue in the alpha-globin gene region compared with the specific DNA of the plasma of the non-pregnant women; counting the proportion of the number of DNA fragments with fetal specific methylation patterns in an alpha-globin gene region and a control region of the plasma DNA of the pregnant woman to the total number of the DNA fragments in the region; the ratio of the number of DNA fragments with a fetal-specific methylation pattern in the α -globin gene region and in the control region to the total number of DNA fragments in the region is compared. In a preferred embodiment, the fetus is tested for alpha-thalassemia by determining dissimilarities in the aforementioned ratios.

In some embodiments, the test sample may be peripheral blood, urine, saliva, sweat of a pregnant woman. In some embodiments, the test sample is peripheral blood of a pregnant woman. Further, the pregnant woman peripheral blood sample is processed to obtain plasma or serum. In some embodiments, the plasma sample is tested. The method belongs to a noninvasive detection means for evaluating the genotype of the alpha-globin gene of a fetus after detecting samples such as peripheral blood, urine, saliva, sweat and the like of a pregnant woman. The test sample can be fetal tissue, such as placental tissue, and testing the sample is an invasive test protocol.

In some embodiments, the sample need not be treated with a DNA methylation state indicator.

In other embodiments, after the test sample is obtained, the sample is treated with a DNA methylation state indicator. In some embodiments, the sample is treated with a DNA methylation state indicator, and methylated and unmethylated modified DNA will show differences. The DNA methylation status indicator can be bisulfite, methylation sensitive restriction enzymes (e.g., HpaII and BstUI), antibodies or binding proteins that recognize methylated DNA, or the like. In some embodiments, the sample is treated with bisulfite.

In some embodiments, the method of detecting the methylation pattern of DNA used in the step of detecting the methylation pattern of DNA in the sample is sequencing. In some embodiments, the sample DNA is constructed as a whole genome bisulfite sequencing library, which is subsequently 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) further comprises real-time PCR, digital PCR, mass spectrometry, single molecule sequencing, and the like.

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 DNA fragments are classified using the methylation pattern. As is well known to the applicants in the art, the statistical analysis may be performed using linkage analysis, one-to-one correspondence analysis, and the like. In other embodiments, machine learning methods can be used to distinguish methylation patterns on free DNA fragments in a test sample.

In some embodiments, the probability that each DNA fragment carrying a particular methylation pattern is derived from the placenta or from the mother is calculated separately. In some embodiments, mathematical techniques can be used to define a DNA fragment as being of fetal origin when the probability of being from a fetus is significantly higher than the probability of being from a mother. As is well known to applicants in the art, this statistical analysis may be performed using techniques well known in the statistical arts such as Chi-Square test, rank-sum test, McDonald's test, hyper-geometry test, ordinary t-test, and the like. In other embodiments, the biological sample can be used as a control comparison, e.g., a DNA fragment aligned to the genome of the α -globin gene region and carrying a particular methylation pattern that is observed only in a maternal peripheral blood free DNA sample carrying a normal fetus and not in a maternal peripheral blood free DNA sample carrying a homozygous α -globin gene region deletion mutant fetus is considered to be the only α -globin gene region from the fetal genome. As is well known to the Applicant, there are many ways of selecting such biological samples, for example, it is possible to select biological samples from the group consisting of maternal peripheral blood, placental samples, placental villus samples, amniotic fluid puncture samples, cord blood samples, etc. Different biological samples were selected without changing the results. After this step is completed, each DNA fragment may be uniquely defined as a "DNA fragment of fetal origin" or "DNA fragment not necessarily of fetal origin".

In some embodiments, analysis counts the number of DNA with fetal-specific methylation patterns, aligned to DNA within the α -globin gene region as well as to control regions (collectively referred to as "genomic regions" below). Specifically, the fraction of fetal-derived DNA fragments to the total number of DNA fragments was analyzed. The ratio should be consistent between different genomic regions on a statistical basis. Within a certain genomic region, a deviation of this ratio from the mean value may indicate a copy number change in this region of the fetal genome. Furthermore, by comparing the ratio of the fetal-derived DNA fragments in the α -globin gene region to the total number of DNA fragments with the ratio of the fetal-derived DNA fragments in the control gene region to the total number of DNA fragments, one can know whether the α -globin gene region of the fetus may have copy number deletion or duplication.

In some embodiments, as is well known to applicants in the art, mutations and genetic defects in genes carried by the fetus may result in altered methylation patterns of other genomic regions (hereinafter "cognate regions"). By comparing the methylation pattern changes of the relevant regions, the mutation status of genes carried by the fetus can also be presumed. In some embodiments, the methylation pattern of the cognate region can be altered to infer whether the fetus carries an alpha-globin gene defect.

The method is suitable for detecting fetal alpha-thalassemia diagnosis, including alpha-globin gene deletion type and mutant type. In some embodiments, the methods are used to diagnose a fetus with a total alpha-globin gene deletion. For example, the fetal α -globin gene is a homozygote formed by a SEA-deleted allele (SEA/SEA). The fetus with the total deletion of the alpha-globin gene shows anemia with general edema, is easy to cause intrauterine death, and can cause serious complications of pregnant women. In some embodiments, the fetal α -globin gene is a genotype formed by a combination of SEA, THAL, α 3.7, α 4.2 deletion alleles, e.g., SEA/SEA, SEA/α 3.7, SEA/α 4.2, and the like. In some embodiments, the fetal α -globin gene carries a point mutation, e.g., α CS, α WS. In addition, the methods can also assess the presence or risk of onset of a pregnancy-associated disorder. For example, the pregnancy-associated disorder is premature birth or the like.

In another aspect of the disclosure, a kit for detecting whether a fetus has alpha-thalassemia is provided. The kit contains reagents required for the enrichment of characteristic free DNA. In some embodiments, the kit may further comprise a DNA methylation status indicator. In some embodiments, the characteristic free DNA is enriched by solution phase hybridization probe capture. In some embodiments, the characteristic source free DNA is enriched by polymerase chain reaction amplification (PCR). In some embodiments, the characteristic source-free DNA is enriched by an anchored nucleic acid amplification method (anchor PCR). In some embodiments, the kit includes reagents that use the enriched characteristic free DNA for sequencing-by-synthesis detection (i.e., so-called pooling reagents). In some embodiments, the kit includes reagents that use free DNA for single molecule sequencing assays (i.e., so-called pooling reagents). In some embodiments, software is also provided for statistical analysis of characteristic free DNA, and the fraction of fetal derived DNA within the α -globin gene region is compared to standard controls to determine the genotype of the fetal α -globin gene locus.

ADVANTAGEOUS EFFECTS OF INVENTION

In one embodiment of the disclosure, the disclosure finds that in the genomic region where the globin gene is located (e.g., the region between 190000-250000 base pairs of human chromosome 16), the fetal DNA has a specific methylation pattern that is different from that of the maternal DNA.

In another embodiment of the present disclosure, the present disclosure provides a number of entirely new, highly fetal-specific DNA methylation markers. The markers can distinguish maternal and fetal DNA at a single molecule level, thereby providing a means for independent analysis of fetal DNA fragments. Therefore, the present disclosure further provides a more accurate, more efficient, and more convenient method for non-invasively diagnosing fetal alpha-thalassemia. The method exhibits a high degree of accuracy in the tests provided by the present disclosure. The present disclosure also provides kits for prenatal diagnosis of fetal alpha-thalassemia.

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 amount of fetal DNA in maternal free DNA, and further be used for the assessment of other fetal disorders as well as pregnancy-related disorders or risks of pregnancy in pregnant women.

Drawings

FIGS. 1A-1C show the fetal-to-maternal differential methylation sites found by whole genome methylation sequencing. Wherein, FIG. 1A shows that there is a steady methylation (black: high; white: low) level difference between each placental sample and the maternal sample. FIG. 1B shows the aforementioned fetal-to-maternal differential methylation sites, present on each chromosome. FIG. 1C shows a plurality of consecutive CpG sites distributed within the same genomic region, in part, among the aforementioned fetal-to-maternal differential methylation sites.

FIG. 2 shows the fetal-to-maternal differential single molecule methylation markers found by whole genome methylation sequencing. Wherein, part A in FIG. 2 shows that at this exemplary genomic position, there is a stable methylation (black: high; white: low) level difference at each CpG site between the placental sample and the maternal sample, while both placental-specific single molecule methylation markers (molecules with white color blocks) and maternal-specific single molecule methylation markers (molecules with black color blocks) can be observed. Part B in FIG. 2 shows that at this exemplary genomic position, there is not necessarily a stable methylation (black: high; white: low) level difference at every CpG site between the placental and maternal samples, but a placenta-specific monomolecular methylation marker (molecules with white color blocks) is still observed

FIG. 3 shows the discovery of fetal-specific methylation markers of the alpha-globin genomic region by whole genome methylation sequencing. Wherein, part A in FIG. 3 shows that there is a specific monomolecular methylation marker (black: high; white: low) difference between the placenta sample and the maternal sample. The DNA molecules are all DNA with demethylation pattern, and can only be found in placenta samples. Part B of figure 3 shows the specific single molecule methylation markers described above, which were only observed in villi with wild-type or heterozygous SEA-deficient genotype carried in the alpha-globin genomic region, but not in villi with homozygous SEA-deficient genotype and peripheral blood of non-pregnant women. The experiment proves that the single-molecule methylation marker can only come from fetal genome, cannot come from maternal genome, and is a specific alpha-globin genome region fetal-derived DNA fragment marker. In particular, the family of single molecule methylation markers is located within the 190000-230000 base pair (GRCh37 version of the genome) human chromosome 16 region. More specifically, the family of single molecule methylation markers, including the combination of CpG sites and methylation status in table 6.

Fig. 4 shows that in 68 pregnant women plasma, pregnant women carrying homozygous SEA-deleted genotype fetuses had significantly lower local fetal-to-fetal ratio of the normalized alpha-globin genomic region than other pregnant women. And (3) scribing according to the normalized local fetal source proportion of 0.01, wherein the sensitivity is 100 percent and the specificity is 96 percent.

FIG. 5 shows the results of plasma DNA detection of pregnant women tested for unknown fetal genotype using placenta-specific methylation patterns.

Detailed description of the preferred embodiments

Definition of

The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification can mean "one," but can also mean "one or more," at least one, "and" one or more than one.

As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Also, the terms "comprising," "having," "including," or "containing" are intended to be inclusive and mean that there may be additional, unrecited elements or method steps.

Throughout this specification, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless expressly indicated to be merely an alternative or a mutual exclusion between alternatives.

A "site" corresponds to a single site, which can be a single base position or a group of related base positions, such as CpG sites.

"DNA methylation" generally refers to the methylation of the 5' carbon of a cytosine residue in a nucleotide, and in the human genome, a significant amount of DNA methylation occurs at cytosine in CpG dinucleotides, C is cytosine, G is guanine, and p is a phosphate group. DNA methylation can also occur in cytosine in CHG and CHH nucleotide sequences, where H is adenine, cytosine or thymine. DNA methylation can also occur on non-cytosines, such as N6-methyladenine. In addition, DNA methylation may be in the form of 5-hydroxymethylcytosine or the like.

"methylation site" refers to a single or multiple base position at which a methylation modification is likely to occur. Such as a CpG site, a CHG site, or a CHH site. In some cases, the methylation sites are equivalent to CpG sites.

"methylation pattern" is a description of the overall methylation status of the sample DNA within the region. Can be "methylation state", "methylation level", "methylation density" or "single molecule methylation state" and combinations thereof.

"methylation state" refers to the characteristic of a particular genomic locus of a segment of DNA that is associated with methylation. Such features include, but are not limited to: whether any cytosine (C) residue within the DNA sequence is methylated, the position of the methylated C residue, the percentage of methylated C at any particular stretch of residues, and allelic differences due to, for example, differences in allelic origin. The term "methylation pattern" or "methylation profile" also refers to the relative or absolute concentration of methylated or unmethylated C of any particular residue extension in a biological sample. For example, if one or more cytosine (C) residues within a DNA sequence are methylated, it may be referred to as "hypermethylation"; if one or more cytosine (C) residues within a DNA sequence are unmethylated, it may be referred to as "hypomethylation". Similarly, a sequence is considered hypermethylated relative to another sequence of a different origin or of a different individual (e.g., relative to maternal nucleic acid) if one or more cytosine (C) residues within the DNA sequence (e.g., fetal nucleic acid) are methylated relative to the other sequence. Alternatively, if one or more cytosine (C) residues within a DNA sequence are not methylated relative to another sequence of a different origin or of a different individual (e.g., mother), then that sequence is considered hypomethylated relative to that other sequence. These sequences are referred to as "differentially methylated", and more specifically, when the methylation status is different between mother and fetus, these sequences are considered "differentially methylated maternal and fetal nucleic acids".

"methylation level" refers to the number of DNA molecules that are methylation modified at the methylation site divided by the number of total DNA molecules containing the site, and is used to describe the overall methylation status of a methylation site in a DNA sample.

"methylation density" is the number of sites within a region that exhibit methylation divided by the total number of reads covering sites in the region.

"Single molecule methylation state" refers to the combination of methylation states of all base sites that a single DNA molecule has that can be methylated. The single molecule methylation state can include 1, two, or more methylation sites. For example, a DNA molecule with two methylation sites may have 4 monomolecular methylation states, including + -, - -and + + ("+" indicates that the methylation site is modified by methylation and "-" indicates that the methylation site is not modified by methylation). In the present disclosure, the single molecule methylation state is used to distinguish maternal and fetal DNA in a plasma sample from a pregnant female. A "single molecule methylation state" can include a methylation state of 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, at least 100, at least 300, at least 400, at least 500, or greater than 500 methylation sites, or a range between any two of the foregoing values.

"DNA methylation status indicator" refers to an agent capable of differentially modifying methylated and unmethylated DNA in the sample.

"detectable marker" refers to a biochemical marker that can mark changes or changes that may occur in the structure or function of systems, organs, tissues, cells and subcellular structures or functions. In one embodiment of the present disclosure, the detection marker is DNA single molecule methylation status.

"sample" refers to any sample derived from a human body and containing DNA molecules. The "sample" for fetal testing may be a peripheral blood sample, a placental villus sample, an amniotic fluid puncture sample, a cord blood sample, a urine sample, a saliva sample, a sweat sample, etc.

"fetus" refers to an embryo 8 weeks after 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 method. In human obstetrics, gestational age refers to the age of the embryo or fetus plus two weeks. In humans, labor usually occurs at gestational age of about 40 weeks, although it is common for labor to occur within 37 to 42 weeks.

The "local fetal-origin ratio" refers to the ratio of fetal-origin DNA fragments to all DNA fragments on the detection region.

"fetal-specific methylation pattern" refers to a pattern of DNA methylation that is present only in placental tissue and not in the plasma of an infertile female.

"alpha-thalassemia" refers to a hereditary hemolytic anemia caused by a defect in the alpha-globin gene. The gene defect of α 0-thalassemia is mainly caused by the deletion of α 1-globin gene. The alpha 5-globin gene cluster is located on chromosome 16 and comprises two highly homologous alpha globin genes of alpha 61 and alpha 72, and both alpha 1 and alpha 2 genes can express alpha globin chains. The normal alpha-globin gene on one chromosome is recorded as (. alpha.2 /), and the genotype of a normal human is (. alpha.3/. alpha.4). Deletion of an alpha-gene on a chromosome is designated as (-alpha /), e.g., (. alpha.) (alpha. /)^3.7/)、(-α^4.2/) and the like. Deletion of two alpha-genes from one chromosome does not allow synthesis of the alpha-globin chain, called alpha⁰Thalassemia, noted (-/-), e.g. (- -^SEA/)、(--^THAIV and (-)^FIL/) etc., which homozygotes show severe α -thalassemia and the affected fetus is prone to intrauterine death. Non-deletion alpha-thalassemia does not generally result in the simultaneous impairment of two alpha-genes on one chromosome, and is therefore referred to as alpha⁺Thalassemia, the allele of which is designated (. alpha.)^MAlpha /) or (alpha)^M/), e.g. (alpha) as is common in China^WSα/)、(α^CSα/)、(α^QSα/)。

The principle of the Whole genome bisulfite sequencing method is that a sample to be detected is treated by using bisulfate, C basic groups which are not methylated in a genome are converted into U, the U is converted into T after PCR amplification, the T is distinguished from C basic groups which originally have methylation modification, and then the T is compared with a reference sequence by combining a high-throughput sequencing technology to judge whether the CpG/CHG/CHH sites are methylated or not, so that the method is particularly suitable for drawing a Whole genome DNA methylation map with single base resolution.

Detection of DNA methylation status

A variety of methods can be used to detect the Methylation state of DNA, such as methylated DNA immunoprecipitation sequencing (MeDIP-Seq, Jacinto et al, 200844 (1), 35-43), simplified representative bisulfite sequencing (RRBS, Meissner et al, Nucleic Acids Res, 2005; 33: 5868-.

In one embodiment, whole genome bisulfite sequencing is capable of analyzing all methylation sites on a genome and can detect a single DNA molecule under single base resolution conditions. Therefore, the method can comprehensively study the DNA methylation state in a genome-wide range and provide a quantitative analysis method of the DNA methylation state. Further, the method can be used for single molecule methylation state assessment.

In recent years, whole Genome bisulfite sequencing has been successfully applied to studies of human epigenetics (Lister et al, Nature,2009,462(7271): 315-. (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 allows for the discrimination of maternal and fetal DNA. Thus, a whole genome bisulfite sequencing method may be selected to implement the methods described in this disclosure.

Identification of alpha-globin Gene-associated fetal DNA markers

In one embodiment, methylation patterns of placental tissue DNA and plasma free DNA from non-pregnant women are detected using whole genome bisulfite sequencing, and the placental tissue DNA is screened for a specific monomolecular methylation state in regions including, but not limited to, the α -globin gene region by analyzing the monomolecular methylation state of the DNA.

In one embodiment, as shown in FIG. 1, the methylation level at a single base site on the genomic region of the α -globin is very noisy and not suitable for single molecule detection. However, by single molecule methylation pattern analysis, placental tissue-derived DNA can be found to be very different from single molecule methylation patterns of plasma free DNA of non-pregnant women. Further, in another embodiment, whole genome bisulfite sequencing is used to determine the genotype as α α α/α α -^SEAAlpha and alpha-^SEA/--^SEAAfter sequencing the placental tissue DNA, the fetus was analyzed for specific monomolecular methylation status within the α -globin gene region. As shown in FIG. 2, in the case of the genotypes α α α/α α -^SEAIn alpha-placental DNA, the DNA in the specific monomolecular methylation state can be detected after the alpha-globin gene is deleted (-one)^SEA/--^SEAGenotype) in which the DNA of said specific monomolecular methylation state cannot be detected in the placental tissue DNA of said genotype. Thus, a DNA fragment of the specific monomolecular methylation state can be used to determine the specific origin of the placental tissue.

Fetal-specific DNA methylation markers

In one embodiment, the present disclosure provides a fetal-specific DNA methylation marker that can be used for prenatal diagnosis of fetal α -thalassemia.

In one embodiment, the marker is selected from the group consisting of single molecule methylation markers between 190000 and 230000 base pairs of human chromosome 16.

In another embodiment, the marker is the level of methylation of a base pair 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 of the genome) or a combination of any two or more of the above base pairs.

In another embodiment, the marker is the level of methylation of a base pair selected from any one of the following positions on human chromosome 16: 229484,229499,229527,229535(GRch37 version of the genome) or a combination of any two or more of the above base pairs.

In another embodiment, the marker is the level of methylation of a base pair selected from any one of the following positions on human chromosome 16: 229484,229499, (GRch37 version of the genome) or a combination of the above base pairs.

In another embodiment, the marker is the level of methylation of a base pair selected from any one of the following positions on human chromosome 16: 229527,229535(GRch37 version of the genome) or combinations of the above base pairs.

In another embodiment, the base pairs are selected from any one of the following positions on human chromosome 16: 229484,229499,229527,229535(GRch37 version of the genome) or a combination of base pairs of any two or more of the above and base pairs selected from one or more of 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(GRch37 version of the genome).

Noninvasive prenatal detection method for alpha-thalassemia

The present disclosure provides a method for non-invasive prenatal diagnosis of fetal alpha-thalassemia that uses a fetal-specific monomolecular methylation state to distinguish fetal DNA from maternal DNA in the blood of a pregnant woman, and then makes statistics on the copy number of the fetal DNA to assess the genotype of the fetal alpha-globin gene.

In one embodiment, the non-invasive prenatal detection method of α -thalassemia to which the present disclosure relates may comprise the steps of: detecting the methylation pattern of DNA in the biological sample; differentiating the different patterns of DNA methylation of total DNA fragments in the biological sample obtained in the detecting step; calculating the methylation pattern of the placenta tissue in the alpha-globin gene region compared with the specific DNA of the plasma of the non-pregnant women; counting the proportion of the number of DNA fragments with fetal specific methylation patterns in an alpha-globin gene region and a control region of the plasma DNA of the pregnant woman to the total number of the DNA fragments in the region; the ratio of the number of DNA fragments with a fetal-specific methylation pattern in the α -globin gene region and in the control region to the total number of DNA fragments in the region is compared. In a preferred embodiment, the fetus is tested for alpha-thalassemia by determining dissimilarities in the aforementioned ratios.

In another embodiment, the present disclosure relates to a method for non-invasive prenatal detection of α -thalassemia, which may comprise the steps of: extracting free DNA in the peripheral blood of the pregnant woman; treating the sample with a DNA methylation state indicator; detecting the methylation pattern of the DNA in the treated sample; counting the number of DNA with fetal specific methylation patterns; analyzing the DNA aligned to the alpha-globin gene region and the control region in the statistical step; comparing the fraction of fetal DNA in the alpha-globin gene region with a standard control to judge the genotype of the fetal alpha-globin gene.

Extraction of free DNA from peripheral blood of pregnant woman

Methods for extracting DNA from plasma are well known to those skilled in the art. The preparation can be carried out according to a conventional method for DNA preparation. In addition, there are also a variety of commercially available kits, such as a column extraction kit from Qiagen, a magnetic bead extraction kit from Life Technologies, and the like. In some embodiments, the DNA in the plasma sample is extracted using a Qiagen column extraction kit. In other embodiments, the DNA in the plasma sample may not be extracted, for example, by direct conversion of the serum sample with bisulfite followed by DNA methylation status detection.

Treating a sample with a DNA methylation status indicator

Whether free DNA is extracted or not, it is preferred that the DNA methylation status indicator treat the sample, e.g., bisulfite. Bisulfite converts unmethylated cytosine to uracil, which remains unchanged, and the converted DNA is subsequently amplified by polymerase chain reaction (Frommer et al ProcNatlAcadSci USA,1992,89:1827-31), and then assayed for conversion to thymine to determine if the cytosine is methylated. The DNA methylation state indicator also comprises methylation sensitive endonuclease, an antibody or a binding protein for identifying the DNA methylation state, an enzyme with DNA oxidant deamination and the like. In other embodiments, the sample may also be treated without a DNA methylation state indicator, for example, by directly detecting the methylation state of a DNA molecule using a nanopore or zero mode waveguide based single molecule sequencing system.

Detecting methylation patterns of DNA in treated samples

Currently, there are a variety of methods available for the detection of DNA methylation patterns. In one embodiment, methods including high throughput sequencing, real-time PCR, digital PCR, mass spectrometry, and microarray can be used for detection of DNA methylation patterns.

Based on the fetal-specific DNA markers provided by the present disclosure, the above methods can be used for prenatal diagnosis of fetal alpha-thalassemia.

The present disclosure can be implemented based on high throughput sequencing platforms, for example, using sequencing platforms such as Miseq, Hiseq, Nextseq, and Novaseq by illumina, Ion Torrent or Ion Proton by Life Technologies, PacBio RS II by pacfic Biosciences system, gridios x5 by Oxford Nanopore Technologies, and the like.

Methylation detection methods based on the principle of bisulfite conversion have become a routine technique for detecting the methylation state of DNA and are well known to those skilled in the art. In one embodiment of the present disclosure, the sample DNA is bisulfite converted using the EZ DNA Methylation-Gold bisulfite conversion kit from Zymo Research.

In a specific embodiment of the present disclosure, the maternal plasma DNA is repaired, added with a, and then ligated with Illumina methylated adaptors, and then the ligation products of the DNA and adaptors are bisulfite converted and then amplified using Polymerase Chain Reaction (PCR) to obtain a sequencing library, which is sequenced on an Illumina Nextseq500 sequencer to obtain the detection result of the methylation state of the maternal plasma DNA in the whole genome range.

In some embodiments, maternal plasma DNA is first bisulfite treated and then a sequencing library is constructed, followed by detection using a high throughput sequencing platform. Various Methods and commercial kits are presently disclosed for constructing sequencing libraries for bisulfite-converted DNA samples, such as random primer amplification-based Methods (Fumihito et al, Nucleic Acids Research,2012,40(17): e136-e 136; Khanna et al, Nature Methods,2013,10(10)), single-stranded nucleotide ligation-based Methods (Raine et al, Nucleic Acids Research,2016,45(6): e 36; Gansuge et al, Nucleic Acids Research,2017,45(10): e79), Terminal deoxynucleotidyl transferase (TdT) based Methods (Pen et al, Nucleic Acids Research, 43(6 e): 35), and the like. In some embodiments, the bisulfite converted DNA sample is subjected to a sequencing library using a commercial kit, such as the Truseq DNA methylation kit from Illumina, the Accel-NGS Methyl-Seq DNAlibrary kit from Swift Biosciences, and the like.

In other embodiments, targeted detection of the methylation state of DNA in a region of interest is accomplished by hybrid capture of the region of interest using a liquid or solid phase probe, followed by sequencing. In one aspect, the probes can be complementary paired to genomic sequences, for example, the sample DNA can be repaired, A can be added, and then ligated to Illumina methylated adaptors, the ligation products of the DNA and adaptors can be incubated with the probes for hybridization, the probes can be washed, bisulfite converted and amplified by Polymerase Chain Reaction (PCR) to obtain a sequencing library, and the sequencing library can be sequenced on an Illumina sequencer to obtain a detection of the methylation state of the sample DNA over the region of interest (HingB, Ramos et al, Epigenetics 2015; 10: 581-96). Alternatively, the probe may be complementarily paired with a bisulfite-converted DNA sequence, for example, the sample DNA may be repaired, added with A, then ligated with Illumina methylated adaptor, followed by bisulfite conversion, and then the amplification product is incubated with the probe for hybridization, after washing the probe, the amplification is performed by Polymerase Chain Reaction (PCR) to obtain a sequencing library, and the sequencing library is sequenced on an Illumina sequencer to obtain the detection result of the methylation state of the sample DNA in the region of interest (Allum et al, Nat Commun, 2015; 6: 7211; Li et al, Nucleic Acids Res 2015; 43: e 81). 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-. In other embodiments, the DNA hybridized to the probe may have been ligated to an adapter (Wang et al, Bmc Genomics,2011,12(1): 597). In some embodiments, the DNA hybridized to the probe may be adaptor-unattached. For example, the DNA is incubated with RNA probes by hybridization, the probes are washed, the RNA probes are degraded with rnases, the captured DNA is library-constructed and then bisulfite-treated, PCR-amplified (or captured DNA is bisulfite-treated and then library-constructed, then PCR-amplified), and the sequencing library is sequenced.

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-. 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 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-. In other embodiments, the DNA methylation status is detected based on methylation sensitive restriction endonuclease sequencing (MRE-seq, Maunakaea et al, Nature.2010; 466: 253-257) that can be selected based on the fetal-specific DNA methylation marker regions provided by the present disclosure.

In some embodiments, the sample DNA is bisulfite treated, and the methylation state of the sample DNA is analyzed by Polymerase Chain Reaction (PCR) amplification of the region of interest, followed by sequencing of the amplified products. In some embodiments, the PCR may be a multiplex PCR. In some embodiments, the PCR amplification is performed using microdroplet techniques (Komori et al, Genome Research,2011,21(10): 1738-.

Single molecule sequencing platforms (e.g., MinION from Oxford Nanopore, PacBio RS II from Pacific Biosciences systems, etc.) will allow direct detection of the methylation status of DNA molecules without bisulfite conversion (including N6-methyladenine, 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 state of sample DNA is analyzed using a single molecule sequencing platform without bisulfite conversion.

In other embodiments, the DNA methylation status can be analyzed using other techniques, such as microarray, real-time PCR, digital real-time PCR, mass spectrometry, and the like (Plongthongkum et al, Nature Reviews Genetics,2014,15(10): 647-61; Consortium et al, Nature Biotechnology,2016,34(7): 726). One of skill in the art will readily recognize that, based on the fetal-specific DNA methylation markers provided by the present disclosure, prenatal diagnosis of fetal α -thalassemia, including but not limited to non-invasive prenatal diagnosis of the fetus, can be achieved using any method that can detect the DNA methylation status.

Analysis of DNA methylation patterns

"methylation pattern" is a description of the overall methylation status of the sample DNA within the region. In some embodiments, the analysis of DNA methylation patterns can be selected from the analysis of DNA methylation status.

In some embodiments, the "analysis of DNA methylation patterns" is a statistic of the number of DNA with fetal-specific methylation patterns.

In some embodiments, the DNA fragment information obtained by the Bisulfite treatment, pooling, sequencing, can be compared to a human reference genome using methods known to those skilled in the art, such as Bismark (Felix Krueger and Simon R.Andrews (2011). Bismark: a flexible alignment and methylation controller for bisult-Seq applications. biologics), bwa-meth (Brent S.Pedersen, et al. (2014). Fast and acquisition alignment of long binary-Seq reads. biologics), and the like, such as: GRCh37(feb.2009, hg 19). In this process, a mismatch of C bases on the reference genome and T bases on the sequenced fragment will be considered as an unmethylated C base, while an unmatched C base will be considered as a methylated C base. The bases on each single-molecule DNA fragment are analyzed to define the single-molecule methylation pattern they carry. Further, DNA fragments with specific methylation patterns are classified as different sources. The classification was based on the unimolecular methylation pattern described previously. After alignment, a bam file was obtained, and the unimolecular methylation pattern of the DNA fragment was extracted using epiread command of the bisguit kit (version: 0.2.0.20161222). The specific monomolecular methylation pattern of the placental genome, which stably existed compared to the non-pregnant female episomal DNA genome, was obtained by MASS (version: 7.3.51.1) analysis, a statistical software package for R language.

In some embodiments, DNA fragments that conform to the fetal-specific DNA methylation patterns provided by the present disclosure can be considered to be DNA fragments of fetal origin. One of skill in the art will readily recognize that prenatal diagnosis of fetal α -thalassemia can be achieved using any reasonable statistical means to classify DNA fragments based on the fetal-specific DNA methylation markers provided by the present disclosure.

In some embodiments, the prenatal diagnosis of fetal α -thalassemia may be selected from a non-invasive prenatal diagnosis of a fetus.

Determining the genotype of the fetal alpha-globin gene

In some embodiments, the "determining the genotype of the fetal α -globin gene" can be the result of an analysis step of DNA methylation patterns, such as counting the number of DNAs with fetal-specific methylation patterns, aligned to DNA in the α -globin gene region and to a control region. Comparing the fraction of fetal DNA in the alpha-globin gene region with a standard control to judge the genotype of the fetal alpha-globin gene.

In some embodiments, the number of DNA fragments of fetal origin on the control genomic region (non a-globin genomic region) and the a-globin genomic region may be counted based on the results of the analysis in the step of analyzing the DNA methylation pattern. Further, the ratio of the fetal-origin DNA fragments to all the DNA fragments in a certain genomic region (hereinafter referred to as "local fetal-origin ratio") is calculated. In some embodiments, the local fetal origin ratio is defined by one single molecule methylation state. In other embodiments, the local fetal origin ratio may be defined by two or more monomolecular methylation states. In some embodiments, the local fetal source ratio is an average of the fetal source ratios corresponding to a single-molecule methylation state, and in other embodiments, the local fetal source ratio is a weighted average of the fetal source ratios corresponding to a single-molecule methylation state. Since the control genomic region is always normal by default, the copy number of the fetal α -globin genomic region can be estimated by comparing the ratio of the local fetal origin ratio of the α -globin genomic region to the local fetal origin ratio of the control genomic region (hereinafter referred to as the relative α -globin gene ratio). In some embodiments, the relative alpha-globin gene ratio of the peripheral blood of the pregnant woman to be tested is only counted to infer the genotype of the carrying fetus. In other embodiments, the method can also be used for sequencing the peripheral blood free DNA of pregnant women carrying fetuses with known alpha-globin genotypes, counting the relative alpha-globin gene proportion, calculating the statistical distribution, and further using mathematical statistical test to presume that the tested pregnant women carry the fetal alpha-globin genotype. One of skill in the art will readily recognize that prenatal diagnosis of fetal α -thalassemia, including but not limited to non-invasive prenatal diagnosis of a fetus, can be achieved using any reasonable statistical means based on the fetal-specific DNA methylation markers provided by the present disclosure.

In another embodiment, the present disclosure relates to a method for non-invasive prenatal detection of α -thalassemia, which may comprise the steps of: obtaining a detection sample; and (4) preprocessing the detection sample.

Test sample acquisition

In one embodiment, the "test sample obtaining" step is a step of obtaining peripheral blood of the pregnant woman.

The method for acquiring the peripheral blood of the pregnant woman comprises the following steps: a biological sample of a pregnant woman containing maternal and fetal DNA is taken, including but not limited to the blood, urine, saliva, etc. of the pregnant woman, preferably a blood sample of the pregnant woman. The blood sample includes, but is not limited to, peripheral blood of a pregnant woman. Peripheral blood is collected from a pregnant woman of suitable gestational age, which may 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 a time between any two of the above time points, preferably 6 weeks to 28 weeks, more preferably 10 weeks to 22 weeks. The peripheral blood of the pregnant woman is collected, transported or stored according to standard procedures adopted by hospitals or institutions. The container for collecting the peripheral blood of the pregnant woman may be a commercially available product such as a Streck Tube blood collection Tube.

Pretreatment of test samples

In one embodiment, the "pretreatment of a test sample" step is a preparation step of peripheral blood plasma of a pregnant woman.

The preparation method of the pregnant woman peripheral blood plasma comprises the following steps: methods for separating plasma or serum from maternal peripheral blood are well known to those skilled in the art. For example, the pregnant woman peripheral blood sample is centrifuged at 1600g to obtain plasma or serum. In a specific embodiment, peripheral blood samples collected from Streck Tube blood collection tubes are centrifuged at 1600g at 4 deg.C, then plasma is transferred to a new centrifuge Tube, and plasma is centrifuged at 16000g at 4 deg.C to remove residual blood cells.

Examples

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, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

All reagents used in this example were commercially available unless otherwise noted.

Example 1: determination of the methylation status of DNA in placental tissue and of free DNA in plasma of non-pregnant women

Subject enrollment and sample Collection

Subjects were enrolled from the prenatal diagnostics department of women's health care institute in Guangdong province. The study and collection of human clinical samples were approved by the institutional review board and informed consent was obtained for each subject. Placental tissue from the early stages of pregnancy was collected immediately after termination of selective pregnancy. Placental tissue from the late gestation period was collected immediately after selective cesarean of simple pregnancy. Peripheral blood from non-pregnant female volunteers was collected (10mL, Streck 21896210 mL Cell-Free DNA BCT non-invasive blood collection tubes).

2 sample treatment

Peripheral blood samples were centrifuged at 1600g for 10 min at 4 ℃ and plasma fractions were centrifuged at 16000g for 10 min at 4 ℃ to further remove residual blood cells.

3DNA extraction

DNA was extracted from placental Tissue using the DNeasy Blood & Tissue kit (manufacturer: QIAGEN; catalog No. 69506) according to the manufacturer's instructions. Free DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid extraction kit (manufacturer: QIAGEN; catalog No.: 55114) according to the manufacturer's instructions.

4 Whole Genome Bisulfite Sequencing (WGBS)

4.1 placental tissue DNA fragmentation treatment

The placenta tissue DNA was disrupted by an ultrasonic disruptor model S220 (manufacturer: Covaris; catalog number: S220) according to the parameters shown in Table 1, to achieve the fragmentation.

TABLE 1 placental tissue DNA fragmentation processing parameters

4.2 ligation of DNA to methylated adaptors

Using the HyperPreP pooling kit, 1. mu.g of fragmented placental tissue DNA or 10-50ng of non-pregnant female free DNA was repaired, A was added, followed by ligation of Illumina methylated Truseq adaptor (primer sequence No.; 001/002). The sources of reagents or kits used in the foregoing steps are shown in table 2A. The sources of reagents or kits used in the foregoing steps are shown in table 2B.

Table 2A sequences of primers used

TABLE 2B sources of reagents or kits in step 1.4.2

DNA repair and purification

The repairing reaction solution was prepared as shown in Table 3, mixed well, and reacted at 20 ℃ for 30 minutes.

TABLE 3 Components and contents of repair reaction solution

Reaction components	Volume (μ L)
		DNA	50
Nuclease-free Water	8
		10X KAPA End Repair Buffer	7
KAPA End Repair Enzyme Mix	5

After the reaction was completed, 120. mu.L of AgencourtAmpur XP magnetic beads incubated at room temperature for 30 minutes were added to purify the DNA. DNA was not eluted from the beads according to the instructions of the HyperPreP pooling kit.

A-labeling and purification of DNA

A-labeling reaction solution of DNA was prepared as shown in Table 4, and after mixing well, the reaction was carried out at 30 ℃ for 30 minutes.

TABLE 4 preparation of A-labeling reaction solution for DNA

Reaction components	Volume (μ L)
		AgencourtAmpure XP magnetic bead (DNA)	-
Nuclease-free Water	42
		10X KAPA A-Tailing Buffer	5
KAPA A-Tailing Enzyme	3

After the reaction was complete, 90. mu.L of PEG/NaCl incubated for 30 minutes at room temperature was added

And purifying the DNA by using a Solution magnetic bead to obtain the DNA with the terminal added with A.

Ligation of DNA to adapters

Ligation reaction solutions were prepared as shown in Table 5, mixed well, and reacted at 20 ℃ for 15 minutes.

TABLE 5 ligation reaction solution preparation

Reaction components	Volume (μ L)
		AgencourtAmpure XP magnetic bead (DNA)	-
Nuclease-free Water	30
		5X KAPA Ligation Buffer	10
Adapters (10. mu.M, 001/002)	5
		KAPA DNA Ligase	5

After the reaction was complete, 50. mu.L of PEG/NaCl incubated for 30 minutes at room temperature was added

And (3) purifying the DNA by using a Solution magnetic bead, and finally adding 20 mu L of nucleic-free Water for elution to obtain the DNA connected with the adaptor.

4.3 bisulfite conversion

The adaptor-ligated DNA was bisulfite-converted using EZ DNA Methylation-Gold bisulfite conversion kit (manufacturer: ZYMO Research; catalog # D5005) according to the manufacturer's instructions and finally eluted using 23. mu.L of nucleic-free Water.

4.4PCR amplification and sequencing library purification

The bisulfite converted DNA was PCR amplified using the KAPA HiFiHotStart Uracil + ReadyMix PCR amplification kit (manufacturer: Roche KapaBiosystems; catalog # KK2802) according to the manufacturer's instructions. After the PCR reaction was completed, DNA was purified using AgencourtAMPure XP magnetic beads to obtain a sequencing library.

4.5 sequencing library quantification and sequencing

Library fragment distribution was detected using Agilent 2100 bioanalyzer (Agilent Technologies, Cat. No. G2939BA) and Agilent high sensitivity DNA kit (Agilent Technologies, Cat. No. 5067-; sequencing library concentrations were determined using a library quantification kit (KAPA Biosystems, cat # KK4824) and DNA quantification standards and a premixed primer kit (KAPA Biosystems, cat # KK4808) according to the manufacturer's instructions.

Sequencing libraries were sequenced using the illuminainnextseq sequencer according to the manufacturer's instructions.

Example 2: identification of fetal-specific methylation pattern markers

Previous studies have shown that fetal free DNA is mainly derived from placental tissue (Papageorgiou et al, Am J Pathol 2009; 174: 1609-.

Further data analysis was performed based on the sequencing results obtained in example 1. The sequenced data were aligned using bwa-meth (Version: 0.2.0). After alignment, the single-molecule methylation pattern of the DNA fragment was extracted by using the epiread command of the biscuit kit (version: 0.2.0.20161222). The specific monomolecular methylation pattern of the placental genome, which stably existed compared to the non-pregnant female episomal DNA genome, was obtained by MASS (version: 7.3.51.1) analysis, a statistical software package for R language.

The experimental results are as follows: on the genome (version GRCh 37), all possible fetal-specific methylation sites present on the whole genome were found, as shown in fig. 1A-1C. These genomic loci, in the fetus, have a large difference in methylation levels from the maternal ones.

Example 3: determination of the methylation status of DNA in placental villus tissue and of free DNA in plasma of non-pregnant women

1. Subject enrollment and sample collection

Subjects were enrolled from the prenatal diagnostics department of women's health center in Guangzhou city. The study and collection of human clinical samples were approved by the institutional review board and informed consent was obtained for each subject. Placenta villi are collected under the guidance of ultrasound in the middle pregnancy (11-14 weeks gestational age). Peripheral blood from non-pregnant female volunteers was collected (10mL, Streck 21896210 mL Cell-Free DNA BCT non-invasive blood collection tubes).

2. Sample processing

Peripheral blood samples were centrifuged at 1600g for 10 min at room temperature and plasma fractions were centrifuged at 16000g for 10 min at room temperature to further remove residual blood cells. .

DNA extraction

DNA was extracted from placental villus Tissue using the DNeasy Blood & Tissue kit (manufacturer: QIAGEN; catalog No. 69506) according to the manufacturer's instructions. Free DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid extraction kit (manufacturer: QIAGEN; catalog No.: 55114) according to the manufacturer's instructions.

4. Whole Genome Bisulfite Sequencing (WGBS)

The method of implementation is the same as the corresponding part in example 1.

5. Analysis after sequencing: the sequenced data were aligned using bwa-meth (Version: 0.2.0). According to The fetal-specific single-molecule methylation markers found in example 2, samtools (Version:1.3.1) { Heng Li et al (2009). The Sequence Alignment/Map format and samtools, bioinformatics } were used to extract all DNA fragments covering The fetal-specific methylation markers. The unimolecular methylation pattern of the above DNA fragment was extracted using the epiread command of the bisguit kit (Version: 0.2.0.20161222). Fragments that fit this pattern methylation marker were obtained by analysis of the statistical software package MASS (Version: 7.3.51.1) in the R language.

The experimental results are as follows: on the genome (version GRCh 37), fetal-specific single-molecule DNA fragment methylation patterns were found near all possible fetal-specific methylation sites present on the whole genome found in example one, as shown in fig. 2 (where part a in fig. 2 and part B in fig. 2 list raw sequencing results, with methylation modifications represented in black, demethylation modifications represented in white, other bases represented in gray, the upper profile being the average methylation modification at each base, and the lower DNA fragment profile being the methylation pattern at each of the raw sequencing-derived fragments). The methylation patterns on these DNA fragments differ greatly in the level of methylation in the fetus from that in the mother. Certain monomolecular methylation patterns are present only in tissues of fetal origin and not in the mother. Therefore, the single-molecule methylation pattern found above can be used to accurately trace the source of each DNA fragment.

Example 4: fetal-specific methylation marker analysis of alpha-globin genomic regions

1. Determination of villus genotype: the Gap-PCR method was used.

The genotype of the fetal villi is detected by utilizing Shenzhen shenshenshentang alpha-thalassemia gene detection kit (Gap-PCR method) (national standard S20060084) according to the instructions of the manufacturer.

2. Analysis after sequencing: the sequenced data were aligned using bwa-meth (Version: 0.2.0). According to The fetal-specific single-molecule methylation markers found in example 2, samtools (Version:1.3.1) { Heng Li et al (2009). The Sequence Alignment/Map format and samtools. bioinformatics } were used to extract all DNA fragments covering The fetal-specific methylation markers. The unimolecular methylation pattern of the above DNA fragment was extracted using the epiread command of the bisguit kit (Version: 0.2.0.20161222). Fragments that fit this pattern methylation marker were obtained by analysis of the statistical software package MASS (Version: 7.3.51.1) in the R language.

The experimental results are as follows: fetal-specific monomolecular methylation patterns found on the genomic (GRCh37 version) α -globin genomic region are shown in part a of fig. 3 and table 6. (FIG. 3 shows the results of the original sequencing in which methylation modifications are shown in black, demethylation modifications are shown in white, other bases are shown in grey, the upper histogram is the mean methylation modification at each base, the lower DNA fragment is shown as the methylation pattern at each fragment obtained from the original sequencing; Table 6 shows the placenta-specific methylation patterns of single molecules in the genomic region of alpha-globin, the first column shows the specific methylation state of a single molecule, where-1 shows the methylation of the site, and 1 shows the methylation modification at the site, the second column shows the specific genomic position information of the methylation state of a single molecule, such as-1, -1/16, 221645,221665 shows the simultaneous demethylation modification of the 221645 th and 221665 th bases of chromosome 16. it is to be noted that, wherein the CpG sites comprise a unimolecular methylation pattern for both the plus and minus strands. ) In particular, we found that specific fetal monomolecular methylation markers were present between the 190000-230000 base pair positions on chromosome 16, as shown in section B of FIG. 3. These single-molecule methylation markers were only observed in placentas with wild-type or heterozygous SEA-deficient genotypes in the alpha-globin genomic region, but not in the peripheral blood of placentas with homozygous SEA-deficient genotypes and non-pregnant women. This experiment argues that the single-molecule methylation markers discovered by the applicant can only be derived from the fetal genome, and not from the maternal genome, and are specific markers for the DNA fragments derived from the alpha-globin genomic region.

TABLE 6 fetal-specific monomolecular methylation patterns (wherein the left column in Table 6 is "specific monomolecular methylation state" and the right column is "specific genomic positional information where the monomolecular methylation state is located")

Example 5: analysis of maternal plasma DNA with known fetal genotypes Using placenta-specific methylation patterns

1. Experiment design: subjects were enrolled from the prenatal diagnostics department of women's health center in Guangzhou city. The study and collection of human clinical samples were approved by the institutional review board and informed consent was obtained for each subject. The collected sample is subjected to amniotic fluid puncture, and the fetal genotype is known (Absence or absence of alpha-globin gene) Peripheral blood samples (10mL, Streck 21896210 mL Cell-Free DNA BCT noninvasive blood collection tubes) of pregnant women (11-14 weeks gestational age).

WGBS methylation sequencing: the procedure is as in the corresponding part of example 1.

3. Statistics of Single molecule methylation patterns

Analysis after sequencing: the sequenced data were aligned using bwa-meth (Version: 0.2.0). Based on The previously discovered fetal-specific single molecule methylation markers on The α -globin genomic region and The control region, all DNA fragments aligned to The target region and The control region were extracted using samtools (Version:1.3.1) { Heng Li et al (2009). The unimolecular methylation pattern of the above DNA fragment was extracted using the epiread command of the bisguit kit (Version: 0.2.0.20161222). Fragments that fit the markers of the fetal specific methylation pattern were obtained by MASS (Version: 7.3.51.1) analysis, a statistical software package in the R language.

Statistical analysis: according to the calculation result of the fetal specific methylation pattern marker, local fetal source proportion is calculated for the alpha-globin genome region and the control region respectively, and normalization processing is carried out on the local fetal source proportion of the alpha-globin genome region according to the local fetal source proportion of the control region.

The experimental results are as follows: the results of the experiment are shown in FIG. 4. Of the 68 pregnant women's plasma, those carrying homozygous SEA-deficient genotyped fetuses had significantly lower local fetal-to-fetal ratios of the normalized alpha-globin genomic regions than those of the other pregnant women.

Example 6: testing maternal plasma DNA with unknown fetal genotype using placenta-specific methylation patterns

1. Experiment design: subjects were enrolled from the prenatal diagnostics department of women's health center in Guangzhou city. The study and collection of human clinical samples were approved by the institutional review board and informed consent was obtained for each subject. Placenta villi are collected under the guidance of ultrasound in the middle pregnancy (11-14 weeks gestational age). Maternal peripheral blood samples (10mL, Streck 21896210 mL Cell-Free DNA BCT non-invasive blood collection tubes) were collected just prior to surgery.

3. Determination of villus genotype: the procedure is as in the corresponding part of example 4.

4. By usingPrediction of fetal genotype by placenta-specific methylation patterns

Analysis after sequencing: the procedure is as in the corresponding part of example 5.

Statistical analysis: according to the calculation result, the local fetal source proportion of the alpha-globin genome region and the control region is respectively calculated, and the local fetal source proportion of the alpha-globin genome region is normalized according to the local fetal source proportion of the control region. Samples with local fetal origin ratios below 0.01 for the normalized alpha-globin genomic regions were defined as pregnant women who were likely to carry a homozygous SEA-deficient genotype fetus (i.e., severe alpha-thalassemia).

As a result: the results of the experiment are shown in table 7 and fig. 5. In the plasma of 141 pregnant women, 50 of the homozygous SEA-deleted fetuses were correctly identified with 99% accuracy (50/51), and 89 of the fetuses carrying heterozygous SEA-deleted genotypes or completely wild-type were correctly identified with 99% accuracy (89/90). The sensitivity was 98.04% and the specificity 98.89%. Among them, one undetected example of the severe thalassemia patient is a placenta-chimeric individual. Therefore, the method can detect whether the fetus of the pregnant woman has severe alpha-thalassemia or not in a non-invasive way before delivery.

TABLE 7 pregnant woman pregnant fetus test results

The above examples of the present disclosure are given for clarity of illustration only and are not intended to limit the embodiments of the present disclosure. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present disclosure should be included in the protection scope of the claims of the present disclosure.

Claims

1. Use of a reagent for detecting a fetal-specific DNA methylation pattern that is a DNA single molecule methylation state in the preparation of a kit for detecting whether a fetus has a genetic disease or is at risk of having a genetic disease; wherein the methylation site of the DNA unimolecular methylation state is selected from the region encoding the alpha-globin gene; the fetal genetic disease is alpha-thalassemia, and the region encoding the alpha-globin gene is selected from any one base pair of the following positions of No. 16 chromosome of human GRch37 version: 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, 210763,210800,210821, 832, 834,210837,210868, 873,210964,210974,213104,213116,213178,213185, 213243, 213200,216200, 216216231, 216483,216503, 216216505, 221505, 221560,221645, 221221221221221221221221709, 221221221221221221221221221740, 22122122122122181740, 2295740, 229554, 229527, 22959; or a combination of any two or more of the base pairs described above.

2. The use of claim 1, wherein the region encoding the α -globin gene comprises at least any one base pair selected from the following positions on chromosome 16 of the human GRch37 version: 229484,229499,229527, 229535; or a combination of any two or more of the base pairs described above.

3. The use of claim 2, wherein the region encoding the α -globin gene comprises at least any one base pair selected from the following positions on chromosome 16 of the human GRch37 version: 229484, 229499; or combinations of the above base pairs.

4. Use according to claim 1, wherein the reagent for detecting a fetal-specific DNA methylation pattern is selected from a DNA methylation status indicator and/or a reagent for detecting the presence of fetal-specific methylation pattern DNA in a biological sample from a pregnant woman.

5. The use according to claim 4, wherein the reagent for detecting the presence of fetal-specific methylation pattern DNA in the biological sample from a pregnant woman is selected from the reagents required for enrichment of characteristic free DNA.

6. Use according to claim 4, wherein the DNA methylation status indicator is selected from an antibody or binding protein recognizing methylated DNA, a bisulfite, an enzyme with DNA catalytic oxidation, an enzyme with DNA deamination or a methylation sensitive enzyme, or a combination thereof.

7. Use according to claim 6, wherein the methylation sensitive enzyme is selected from methylation sensitive restriction endonucleases.

8. The use according to claim 7, wherein the methylation sensitive restriction enzyme is selected from HpaII or BstUI, or a combination thereof.

9. The use according to claim 5, wherein the reagents required for enriching for characteristic free DNA are selected from reagents required for a solution phase hybridization probe capture method, reagents required for a polymerase chain reaction amplification method, reagents required for an anchored nucleic acid amplification method, reagents for sequencing-by-synthesis detection, or reagents for single molecule sequencing, or a combination thereof.

10. The use of claim 4, wherein the sample is selected from whole blood, plasma, serum, urine, saliva, sweat, placenta, placental villi, amniotic fluid, or cord blood, or a combination thereof.

11. Use according to claim 1, wherein the test is a prenatal test.

12. The use of claim 11, wherein the prenatal detection is a non-invasive prenatal detection.

13. A detection apparatus for detecting whether a fetus has a genetic disease or is at risk of having a genetic disease, comprising:

a processor;

a memory for storing execution instructions;

wherein the processor, when executing the instructions, implements a method of detecting whether a fetus has a genetic disease or is at risk of having a genetic disease, wherein the method comprises the steps of:

the genetic disease is alpha-thalassemia, the gene region corresponding to the genetic disease is selected from a region coding for alpha-globin, and the region coding for the alpha-globin is selected from any one base pair of the following positions of No. 16 chromosome of a human GRch37 version: 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, 210763,210800,210821, 832, 834,210837,210868, 873,210964,210974,213104,213116,213178,213185, 213243, 213200,216200, 216216231, 216483,216503, 216216505, 221505, 221560,221645, 221221221221221221221221709, 221221221221221221221221221740, 22122122122122181740, 2295740, 229554, 229527, 22959; or a combination of any two or more of the base pairs described above.

14. The detection apparatus of claim 13, wherein the method further comprises the step of determining:

15. The detection device of claim 13, wherein the detection step is preceded by a processing step of: the biological sample is treated with a DNA methylation state indicator.

16. The detection apparatus according to claim 13, wherein in the calculation step, if a certain ratio is aligned to a gene region corresponding to a genetic disease and a DNA fragment carrying a specific DNA methylation pattern can only be observed in a biological sample of a pregnant woman carrying a normal fetus and cannot be observed from a biological sample of a pregnant woman carrying a fetus with a deletion-type genetic disease, the DNA fragment carrying the specific DNA methylation pattern can only be of fetal origin.

17. The detection apparatus according to claim 13, wherein the region encoding α -globin gene comprises at least any one base pair selected from the following positions in human GRch37 version genome No. 16: 229484,229499,229527, 229535; or a combination of any two or more of the base pairs described above.

18. The detection apparatus according to claim 17, wherein the region encoding α -globin gene comprises at least any one base pair selected from the following positions in human GRch37 version genome No. 16: 229484, 229499; or combinations of the above base pairs.

19. The test device of claim 15, wherein the DNA methylation status indicator is selected from an antibody or binding protein that recognizes methylated DNA, a bisulfite, an enzyme having DNA catalytic oxidation, an enzyme having DNA deamination, or a methylation sensitive enzyme, or a combination thereof.

20. A test device according to claim 19, wherein the methylation sensitive enzyme is selected from methylation sensitive restriction endonucleases.

21. The test device of claim 20, wherein the methylation sensitive restriction enzyme is selected from HpaII or BstUI, or a combination thereof.

22. The test device of claim 13, wherein the reagents required to detect the DNA methylation pattern of total DNA fragments in the processed biological sample are selected from the group consisting of reagents required for a liquid phase hybridization probe capture method, reagents required for a polymerase chain reaction amplification method, reagents required for an anchored nucleic acid amplification method, reagents for sequencing-by-synthesis detection, or reagents for single molecule sequencing, or a combination thereof.

23. A non-transitory computer readable storage medium for detecting whether a fetus has or is at risk of having a genetic disease, having stored thereon computer program instructions which, when executed by a processor, implement a method of detecting whether a fetus has or is at risk of having a genetic disease, wherein the method comprises the steps of:

24. The non-transitory computer readable storage medium of claim 23, wherein the method further comprises the step of determining:

25. The non-transitory computer readable storage medium of claim 23, wherein the detecting step, prior to, comprises the processing step of: the biological sample is treated with a DNA methylation state indicator.

26. The non-transitory computer readable storage medium of claim 23, wherein in the calculating step, the DNA fragments of the specific DNA methylation pattern are of fetal origin only if a certain alignment is to a gene region corresponding to a genetic disease and the DNA fragments carrying the specific DNA methylation pattern can only be observed in a biological sample of a pregnant woman carrying a normal fetus and cannot be observed from a biological sample of a pregnant woman carrying a fetus with a deletion-type genetic disease.

27. The non-transitory computer readable storage medium of claim 23, wherein the region encoding an a-globin gene comprises at least any one base pair selected from the following positions in human GRch37 version genome chromosome 16: 229484,229499,229527, 229535; or a combination of any two or more of the base pairs described above.

28. The non-transitory computer readable storage medium of claim 27, wherein the region encoding an a-globin gene comprises at least any one base pair selected from the following positions in human GRch37 version genome chromosome 16: 229484, 229499; or combinations of the above base pairs.

29. The non-transitory computer readable storage medium of claim 25, wherein the DNA methylation state indicator is selected from an antibody or binding protein that recognizes methylated DNA, a bisulfite, an enzyme with DNA catalytic oxidation, an enzyme with DNA deamination, or a methylation sensitive enzyme, or a combination thereof.

30. The non-transitory computer readable storage medium of claim 29, wherein the methylation sensitive enzyme is selected from a methylation sensitive restriction enzyme.

31. The non-transitory computer readable storage medium of claim 30, wherein the methylation sensitive restriction enzyme is selected from HpaII or BstUI, or a combination thereof.

32. The non-transitory computer readable storage medium of claim 23, wherein the reagents required to detect the DNA methylation pattern of total DNA fragments in the processed biological sample are selected from the group consisting of reagents required for a liquid phase hybridization probe capture method, reagents required for a polymerase chain reaction amplification method, reagents required for an anchored nucleic acid amplification method, reagents for sequencing-by-synthesis detection, or reagents for single molecule sequencing, or a combination thereof.