DNA methylation markers in acute lymphoblastic leukemia and their applications

Abstract

The present invention relates to a DNA methylation marker for acute lymphoblastic leukemia and an application thereof. The marker comprises genes ARHGEF3, ATP7B, C14orf102, CoL2A1, LCP2, MAD1L1, MAPK1, LOC100134868, HTR7, BANP, LDLRAD4, NCAM2, PALM2-AKAP2, RRBP1 and SPPL2B, as well as PFKP, PRDM8, PRDM16 or a combination thereof. DNA fragments of the above genes, when used alone or in combination, can be used for detection, diagnosis, classification or prediction, treatment monitoring, prognosis or other evaluation of acute lymphoblastic leukemia. Moreover, the inventors further found 24 more representative DNA fragments related to acute lymphoblastic leukemia among these 18 genes. Their methylation levels were highly correlated with the diagnosis, remission and relapse of acute lymphoblastic leukemia. It was found that the above genomic fragments or their combinations can serve as diagnostic markers for ALL and can effectively distinguish between ALL remission and relapse patients, indicating that they can further serve as screening markers for monitoring minimal residual disease in ALL patients after treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an acute lymphoblastic leukemia DNA methylation marker and application thereof. Background Art

[0002] Acute lymphoblastic leukemia (ALL) is a malignant clonal tumor caused by the abnormal proliferation of lymphoblasts. It is the most common cancer in children, accounting for 26% of all childhood cancers and 75%-80% of childhood acute leukemias. B-cell ALL, comprising 80% of all childhood ALL, is the most common type. The incidence of ALL in adults is lower, accounting for approximately 20% of adult acute leukemias.

[0003] Under normal circumstances, blood stem cells, also known as immune cells, produced in the bone marrow will differentiate into mature blood cells. Blood stem cells can develop into two types: myeloid stem cells and lymphoid stem cells. Myeloid stem cells then develop into three types of mature blood cells: red blood cells (RBCs), which carry oxygen and other substrates to various organs and tissues throughout the body; platelets, which function in blood clotting; and granulocytes (WBCs), which fight infection and disease. Abnormalities in myeloid stem cells are the primary cause of acute myeloid leukemia (AML). Lymphoid stem cells first differentiate into lymphoblasts, which then develop into B lymphocytes, which produce antibodies to defend against infection; T lymphocytes, which assist B lymphocytes in producing antibodies; and natural killer cells, which attack tumor cells and viruses. In patients with ALL, a large number of stem cells transform into lymphoblasts, B lymphocytes, or T lymphocytes, also known as leukemic cells. These cells are unable to effectively defend against infection, leading to a decrease in the number of white blood cells, red blood cells, and platelets in the blood and bone marrow, potentially causing infection, anemia, or bleeding. For most cancers, early detection simplifies subsequent clinical treatment and increases patient benefit. However, to date, there are no specific early detection methods for acute lymphoblastic leukemia. Conventional leukemia testing involves microscopic examination of bone marrow or blood samples, identifying and classifying white blood cells based on their size, shape, and other characteristics. The most important indicator is the maturity of the blood cells. A definitive diagnosis of ALL typically requires that at least 20% of the cells in the bone marrow are immature. Normally, immature cells do not exceed 5% of the bone marrow cells.

[0004] On the other hand, for patients with relapsed and refractory leukemia, there are problems such as difficult treatment monitoring and difficult prognosis. Relapse treatment is very likely to produce drug resistance, and the treatment effect gradually decreases. The detection of minimal residual disease (MRD) after the patient's medication is relieved has become a hot topic in the clinical treatment and detection of different tumors. After leukemia patients achieve complete remission (CR) after treatment, although obvious leukemia cells cannot be detected by conventional morphological methods (observing the bone marrow under a microscope with 100 times magnification), a small amount of leukemia residual cells may still remain in the body. Even if flow cytometry is used for detection, the sensitivity can reach 10 -4 -10 -5 (That is, 1 leukemia cell can be found in 10,000 to 100,000 cells.) However, it is unable to detect earlier cells, before morphological changes occur. Molecular diagnostic methods can detect leukemia cells at an earlier stage, harboring genetic mutations or differences in gene expression. Currently, molecular markers used in clinical testing primarily focus on cytogenetic abnormalities, such as fusion genes and point mutations. However, patients with these cytogenetic and mutational abnormalities only account for a fraction of the disease population. Given the complexity of leukemia, the search for molecular markers that can be used for accurate diagnosis and prognostic stratification is of great significance.

[0005] Numerous studies at home and abroad have demonstrated that abnormal regulation of DNA methylation is involved in the development, progression, and prognosis of tumors. Plasma free DNA methylation is not only closely associated with early tumorigenesis but can also distinguish tissue origin (PMID: 29035356, PMID: 26392541). Combinations of different gene DNA methylation molecular markers have also been applied to tumor screening, particularly for the early diagnosis of pan-cancer diseases (CN 111742062A, CN 109680060A). In the field of hematological diseases, a published invention patent (CN111647661A) established a method for assisting the diagnosis of acute myeloid leukemia by using methylation information from a set of specific gene elements: CYP26C1-DMR, DGKG-DMR, WT1-DMR, and TRIM40-DMR. The authors found that methylation levels in AML patients were significantly higher than those in normal subjects, and the p-value of the logistic regression model was used to determine whether the patient had developed the disease. In addition, published patent applications (CN111411157A) and (CN109852672A) have demonstrated, through genome-wide methylation and genome-wide CpG site methylation capture sequencing, that DNA methylation is specific for the prognosis of juvenile myelomonocytic leukemia and acute myeloid leukemia, respectively. However, the identification of DNA methylation markers for the diagnosis, treatment, and minimal residual disease (MRD) monitoring of acute lymphoblastic leukemia is of great clinical significance, yet research remains limited. Summary of the Invention

[0006] Based on this, one of the objectives of the present invention is to provide a DNA methylation marker or a combination thereof for detecting acute lymphoblastic leukemia.

[0007] The technical solutions for achieving the above objectives are as follows:

[0008] A DNA methylation marker or combination thereof for detecting acute lymphoblastic leukemia, characterized in that the DNA methylation marker is selected from at least one of genes ARHGEF3, ATP7B, C14orf102, CoL2A1, LCP2, MAD1L1, MAPK1, LOC100134868, HTR7, BANP, LDLRAD4, NCAM2, PALM2-AKAP2, RRBP1 and SPPL2B, PFKP, PRDM8, and PRDM16; the sequence of ARHGEF3 is shown in SEQ ID NO.1 or its complementary sequence, the sequence of ATP7B is shown in SEQ ID NO.2 or its complementary sequence, the sequence of C14orf102 is shown in SEQ ID NO.4 or its complementary sequence, the sequence of LDLRAD4 is shown in any one of SEQ ID NO.5-SEQ ID NO.6 or SEQ ID NO.5-SEQ ID NO.6 complementary sequence, the sequence of CoL2A1 is shown in SEQ ID NO. NO.7, the sequence of HTR7 is shown in SEQ ID NO.8, the sequence of LCP2 is shown in SEQ ID NO.9 or its complementary sequence, the sequence of NCAM2 is shown in SEQ ID NO.15 or its complementary sequence, the sequence of BANP is shown in SEQ ID NO.3 or its complementary sequence, the sequence of LOC100134868 is shown in SEQ ID NO.10 or its complementary sequence, the sequence of MAD1L1 is shown in SEQ ID NO.11 or its complementary sequence, the sequence of MAPK1 is shown in any one of SEQ ID NO.12 to SEQ ID NO.14 or the complementary sequence of SEQ ID NO.12 to SEQ ID NO.14, the sequence of PALM2-AKAP2 is shown in any one of SEQ ID NO.16 to SEQ ID NO.17 or the complementary sequence of SEQ ID NO.16 to SEQ ID NO.17, the sequence of RRBP1 is shown in SEQ ID NO.22 or its complementary sequence, and the sequence of SPPL2B is shown in SEQ ID NO.23 to SEQ ID NO.24 or SEQ ID NO.23-SEQ ID NO.24 complementary sequence; the sequence of the PFKP is shown as SEQ ID NO.18 or its complementary sequence; the sequence of the PRDM16 is shown as any one of SEQ ID NO.19-SEQ ID NO.20 or SEQ ID NO.19-SEQ ID NO.20 complementary sequence; the sequence of the PRDM8 is shown as SEQ ID NO.21 or its complementary sequence.

[0009] In some embodiments, the above-mentioned DNA methylation marker or combination thereof is characterized in that the DNA methylation marker combination includes any at least one selected from SEQ ID NO.2 or its complementary sequence, SEQ ID NO.18 or its complementary sequence.

[0010] In some embodiments, the above-mentioned DNA methylation marker or combination thereof is characterized in that the DNA methylation marker combination includes any at least one selected from SEQ ID NO.3 or its complementary sequence, SEQ ID NO.21 or its complementary sequence.

[0011] In some embodiments, the above-mentioned DNA methylation marker or combination thereof is characterized in that the DNA methylation marker combination includes a sequence selected from SEQ ID NO.1-SEQ ID NO.2 or a completely complementary sequence of SEQ ID NO.1-SEQ ID NO.2, a completely complementary sequence of SEQ ID NO.4-SEQ ID NO.9 or a completely complementary sequence of SEQ ID NO.4-SEQ ID NO.9, SEQ ID NO.15 or a complementary sequence thereof, and SEQ ID NO.18 or a complementary sequence thereof;

[0012] And / or, the above-mentioned DNA methylation marker combination includes a sequence selected from SEQ ID NO.3 or its complementary sequence, SEQ ID NO.10-SEQ ID NO.14 or the fully complementary sequence of SEQ ID NO.10-SEQ ID NO.14, SEQ ID NO.16-SEQID NO.17 or the fully complementary sequence of SEQ ID NO.16-SEQ ID NO.17, and SEQ ID NO.19-SEQ ID NO.24 or the fully complementary sequence of SEQ ID NO.19-SEQ ID NO.24.

[0013] In some embodiments, the above-mentioned DNA methylation marker combination includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.24 or the completely complementary sequences of SEQ ID NO.1 to SEQ ID NO.24.

[0014] One of the purposes of the present invention is to provide a use of the above-mentioned DNA methylation markers or a combination thereof in the preparation of a kit for detecting, classifying or predicting, monitoring treatment, prognosing or otherwise evaluating acute lymphoblastic leukemia.

[0015] Another object of the present invention is to provide a detection kit for acute lymphoblastic leukemia.

[0016] The technical solutions for achieving the above objectives are as follows:

[0017] An acute lymphoblastic leukemia detection kit comprises a reagent for detecting the methylation degree of the above-mentioned DNA methylation marker or a combination thereof.

[0018] In some embodiments, the above-mentioned kit is a kit used for detection using polymerase chain reaction technology, in situ hybridization technology, enzymatic mutation detection technology, chemical cleavage mismatch technology, mass spectrometry analysis technology, gene chip technology or gene sequencing technology or a combination thereof.

[0019] In some of the embodiments, the detection method is methylation-specific PCR, sulfite PCR sequencing, real-time quantitative methylation-specific PCR, etc.; high-throughput detection technologies include simplified genome methylation sequencing, whole genome methylation sequencing, DNA enrichment sequencing, pyrophosphate sequencing, sulfite conversion sequencing, etc.; detection technologies based on mass spectrometry-based detection platforms such as GC-MS, LC-MS, MALDI-TOFMS, FT-MS, ICP-MS, SIMS, etc.; and chip-based detection platforms such as 450K and 850K methylation detection technologies.

[0020] In some embodiments, the detection kit includes a capture probe for each DNA methylation marker, wherein the capture probe is: SEQ ID NO.25 and SEQ ID NO.49 for SEQ ID NO.1, and / or SEQ ID NO.26 and SEQ ID NO.50 for SEQ ID NO.2, and / or SEQ ID NO.36 and SEQ ID NO.60 for SEQ ID NO.3, and / or SEQ ID NO.27 and SEQ ID NO.51 for SEQ ID NO.4, and / or SEQ ID NO.37 and SEQ ID NO.61 for SEQ ID NO.5, and / or SEQ ID NO.38 and SEQ ID NO.62 for SEQ ID NO.6, and / or SEQ ID NO.28 and SEQ ID NO.52 for SEQ ID NO.7, and / or SEQ ID NO.35 and SEQ ID NO.59 for SEQ ID NO.8, and / or SEQ ID NO.29 and SEQ ID NO.53 for SEQ ID NO.9, and / or SEQ ID NO. SEQ ID NO.34 and SEQ ID NO.58 for SEQ ID NO.10, and / or SEQ ID NO.30 and SEQ ID NO.54 for SEQ ID NO.11, and / or SEQ ID NO.31 and SEQ ID NO.55 for SEQ ID NO.12, and / or SEQ ID NO.32 and SEQ ID NO.56 for SEQ ID NO.13, and / or SEQ ID NO.33 and SEQ ID NO.57 for SEQ ID NO.14, and / or SEQ ID NO.39 and SEQ ID NO.63 for SEQ ID NO.15, and / or SEQ ID NO.40 and SEQ ID NO.64 for SEQ ID NO.16, and / or SEQ ID NO.41 and SEQ ID NO.65 for SEQ ID NO.17, and / or SEQ ID NO.46 and SEQ ID NO.70 for SEQ ID NO.22, and / or SEQ ID NO.47 and SEQ ID NO.48 for SEQ ID NO.23. NO.71, and / or SEQ ID NO.48 and SEQ ID NO.72 for SEQ ID NO.24.

[0021] In some embodiments, the detection kit includes capture probes for each DNA methylation marker, the capture probes including: SEQ ID NO.25 and SEQ ID NO.49 for SEQ ID NO.1, SEQ ID NO.26 and SEQ ID NO.50 for SEQ ID NO.2, SEQ ID NO.36 and SEQ ID NO.60 for SEQ ID NO.3, SEQ ID NO.27 and SEQ ID NO.51 for SEQ ID NO.4, SEQ ID NO.37 and SEQ ID NO.61 for SEQ ID NO.5, SEQ ID NO.38 and SEQ ID NO.62 for SEQ ID NO.6, SEQ ID NO.28 and SEQ ID NO.52 for SEQ ID NO.7, SEQ ID NO.35 and SEQ ID NO.59 for SEQ ID NO.8, SEQ ID NO.29 and SEQ ID NO.53 for SEQ ID NO.9, SEQ ID NO.34 and SEQ ID NO.58 for SEQ ID NO.10, and SEQ ID NO.35 and SEQ ID NO.59 for SEQ ID NO.9. SEQ ID NO.30 and SEQ ID NO.54 for SEQ ID NO.11 and SEQ ID NO.31 and SEQ ID NO.55 for SEQ ID NO.12 and SEQ ID NO.32 and SEQ ID NO.56 for SEQ ID NO.13, and SEQ ID NO.33 and SEQ ID NO.57 for SEQ ID NO.14 and SEQ ID NO.39 and SEQ ID NO.63 for SEQ ID NO.15 and SEQ ID NO.40 and SEQ ID NO.64 for SEQ ID NO.16 and SEQ ID NO.41 and SEQ ID NO.65 for SEQ ID NO.17 and SEQ ID NO.42 and SEQ ID NO.66 for SEQ ID NO.18 and SEQ ID NO.44 and SEQ ID NO.68 for SEQ ID NO.19 and SEQ ID NO.45 and SEQ ID NO.69 for SEQ ID NO.20 and SEQ ID NO.43 and SEQ ID NO.67 for SEQ ID NO.21 and SEQ ID NO. SEQ ID NO.46 and SEQ ID NO.70 for SEQ ID NO.22 and SEQ ID NO.47 and SEQ ID NO.71 for SEQ ID NO.23 and24 of SEQ ID NO.48 and SEQ ID NO.72.

[0022] Another object of the present invention is to provide a method for detecting acute lymphoblastic leukemia.

[0023] The technical solutions for achieving the above objectives are as follows:

[0024] A method for detecting acute lymphoblastic leukemia comprises the following steps:

[0025] Extracting genomic DNA from the biological sample to be tested;

[0026] The above DNA was subjected to bisulfite conversion;

[0027] Detection of the methylation level of the above-mentioned DNA methylation markers or a combination thereof.

[0028] In some embodiments, the detection of the methylation level of the above-mentioned DNA methylation markers or a combination thereof includes: extracting methylation information of the above-mentioned DNA methylation markers, constructing a library for their amplified products; and evaluating methylation of sequencing data.

[0029] In some embodiments, the above-mentioned DNA methylation marker detection method includes but is not limited to the following technologies: polymerase chain reaction technology, in situ hybridization technology, enzymatic mutation detection technology, chemical cleavage mismatch technology, mass spectrometry analysis technology, gene chip technology or gene sequencing technology.

[0030] In some of the embodiments, the detection method is methylation-specific PCR, sulfite PCR sequencing, real-time quantitative methylation-specific PCR, etc.; high-throughput detection technologies include simplified genome methylation sequencing, whole genome methylation sequencing, DNA enrichment sequencing, pyrophosphate sequencing, sulfite conversion sequencing, etc.; detection technologies based on mass spectrometry-based detection platforms such as GC-MS, LC-MS, MALDI-TOFMS, FT-MS, ICP-MS, SIMS, etc.; and chip-based detection platforms such as 450K and 850K methylation detection technologies.

[0031] In some embodiments, the biological sample is blood, plasma, saliva, or serum.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The inventors have discovered DNA methylation markers highly associated with acute lymphoblastic leukemia (ALL). These markers include 24 DNA fragments from 18 genes: ARHGEF3, ATP7B, C14orf102, CoL2A1, LCP2, MAD1L1, MAPK1, LOC100134868, HTR7, BANP, LDLRAD4, NCAM2, PALM2-AKAP2, RRBP1, and SPPL2B, as well as PFKP, PRDM8, and PRDM16. Combinations of these DNA fragments can be used for the detection, diagnosis, classification, or prediction, treatment monitoring, prognosis, or other evaluation of ALL. Furthermore, the inventors discovered that the methylation levels of these DNA fragments are highly correlated with the diagnosis, remission, and relapse of ALL. By studying the differences in methylation modifications of these gene fragments between ALL patients and healthy controls, the inventors discovered that these genomic fragments or combinations thereof can serve as diagnostic markers for ALL. Furthermore, by detecting the above genomic fragment combination in patients who have achieved remission after ALL treatment and patients who have relapsed after ALL treatment, it is possible to effectively distinguish between patients who have achieved remission and those who have relapsed, indicating that it can be further used as a screening marker for monitoring minimal residual disease (MRD) in patients with ALL after treatment.

[0034] The present invention jointly analyzes the co-methylation characteristics of multiple methylated cytosines on at least one genomic fragment as a biomarker for determining the onset of ALL. It shows significant differences in methylation modifications in clinical samples and is of great significance for the prediction of ALL, targeted drug development and companion diagnosis, especially for screening and diagnosis of minimal residual disease (MRD) after treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The figure shows the distribution of methylation of ATP7B, PFKP, BANP and PRDM8 genes in the disease and control groups in Example 1, where the boxed area is the differentially methylated area, the solid line is the control group, and the dotted line is the simulated methylation level curve of the disease group.

[0036] Figure 2 This is a diagram of the methylation levels of the 18 gene markers in Example 3 in patients with remission and relapse, where Group A is the remission sample group and Group B is the relapse sample group.

[0037] Figure 3 This is the principal component analysis diagram of the marker methylation sequencing data of the remission and relapse patients in Example 3, where Group A is the remission sample group and Group B is the relapse sample group. The horizontal and vertical axes represent the two dimensions after dimensionality reduction, and the percentages in the brackets represent the explanatory power or effectiveness. DETAILED DESCRIPTION

[0038] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the examples, and preferred embodiments of the present invention are provided below. However, the present invention can be implemented in many different forms and is not limited to the examples described herein. The purpose of providing these examples is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. It should be understood that the experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. The various commonly used reagents used in the examples are all commercially available products.

[0039] Definitions To facilitate understanding of this technology, certain terms and phrases are defined below.

[0040] Throughout the specification and claims, the following terms have the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used herein does not necessarily refer to the same embodiment, although it may be. Furthermore, the phrase "in another embodiment" as used herein does not necessarily refer to a different embodiment, although it may be. Thus, as described below, the various embodiments of the present invention can be readily combined without departing from the scope or spirit of the invention.

[0041] Furthermore, as used herein, the term "or" is inclusive and equivalent to the term "and / or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0042] The terms "complementary" and "complementarity" refer to a nucleotide (e.g., a single nucleotide) or polynucleotide (e.g., a sequence of nucleotides) relative to the base pairing rules. For example, the sequence 5'-AGT-3' is complementary to the sequence 3'-TCA-5'. Complementarity can be "partial," where only some of the nucleic acid bases are matched according to the base pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid chains affects the efficiency and strength of hybridization between nucleic acid chains. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids.

[0043] The term "polymerase chain reaction" is used to amplify a target sequence. This method consists of the following steps: introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycles in the presence of a DNA polymerase. The two primers complement the corresponding strands of the double-stranded target sequence. To perform amplification, the mixture is denatured, and the primers are then annealed to their complementary sequences within the target molecule. After annealing, the primers are amplified with a polymerase, forming a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated multiple times (i.e., denaturation, annealing, and extension constitute one "cycle"; there can be many "cycles") to obtain a high concentration of amplified fragments of the desired target sequence. The length of the amplified fragments of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore this length is a controllable parameter. Due to the repetitive nature of this method, it is referred to as the "polymerase chain reaction" ("PCR"). Because the desired amplified fragment of the target sequence becomes the predominant sequence (in terms of concentration) in the mixture, it is said to be "PCR amplified" and is a "PCR product" or "amplicon."

[0044] As used herein, the term "nucleic acid detection assay" refers to any method for determining the nucleotide composition of a target nucleic acid. Nucleic acid detection assays include, but are not limited to, DNA sequencing methods and probe hybridization methods.

[0045] The term "amplifiable nucleic acid" refers to a nucleic acid that can be amplified by any amplification method. It is expected that the "amplifiable nucleic acid" will typically comprise a "sample template."

[0046] The term "sample template" refers to nucleic acids derived from a sample that are used to analyze the presence of a "target" (defined below). In contrast, "background template" is used to refer to nucleic acids other than the sample template, which may or may not be present in the sample. Background template is usually unintentional. This may be the result of legacy, or it may be due to the presence of nucleic acid contaminants that are attempted to be purified from the sample. For example, nucleic acids other than the nucleic acids to be detected from an organism may exist as background for the test sample.

[0047] As used herein, "methylation" refers to methylation of cytosine at positions C5 or N4 of cytosine, N6 of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is typically unmethylated because typical in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" may also refer to amplified DNA that is unmethylated or methylated, respectively, from the original template.

[0048] Thus, as used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, wherein the methyl moiety is not present in recognized typical nucleotide bases. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at the 5-position of its pyrimidine ring. Thus, cytosine is not a methylated nucleotide, and 5-methylcytosine is a methylated nucleotide. In another example, thymine contains a methyl moiety at the 5-position of its pyrimidine ring; however, for the purposes of this article, thymine is not considered a methylated nucleotide when present in DNA, as thymine is a typical nucleotide base of DNA.

[0049] The methylation status can optionally be represented or indicated by a "methylation value" (e.g., representing a methylation frequency, fraction, ratio, percentage, etc.). The methylation value can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing amplification spectra after a bisulfite reaction, or by comparing the sequences of bisulfite-treated and untreated nucleic acids. Therefore, a value such as a methylation value represents the methylation status and can therefore be used as a quantitative indicator of the methylation status in multiple copies of a locus. The degree of co-methylation is represented or indicated by the methylation status of more than one methylation site, and co-methylation is defined as when the methylation status of more than one methylation site within a methylated region is all methylated.

[0050] As used herein, the term "bisulfite reagent" refers to a reagent that, in some embodiments, comprises bisulfite, disulfite, hydrogen sulfite, or a combination thereof. When DNA is treated with a bisulfite reagent, unmethylated cytosine nucleotides are converted to uracil, while methylated cytosine and other bases remain unchanged. Therefore, for example, methylated and unmethylated cytidine in CpG dinucleotide sequences can be distinguished.

[0051] The term "methylation assay" refers to any assay used to determine the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.

[0052] The present invention is further described in detail below with reference to specific embodiments.

[0053] Example 1 Whole-genome methylation sequencing to identify molecular markers

[0054] Ten clinical samples were collected from five pairs of identical twins and divided into two groups. Group 1 consisted of healthy twins serving as the control group, while Group 2 consisted of patients with ALL who had undergone clinical treatment and were in remission. Genomic DNA was extracted from each sample using Tiangen's Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304-03). DNA concentration was quantified using the Qubit fluorescence assay.

[0055] 1 μg of genomic DNA was randomly sheared by physical ultrasound to fragment the DNA into about 300 bp. After purification, the DNA was repaired in the presence of DNA polymerase (15 U of T4 DNA Polymerase and 5 U of Klenow Fragment), polynucleotide kinase (50 U of T4 Polynucleotide Kinase) and 0.4 mM dNTP in a 100 μl system at 20 ° C for 30 min. Subsequently, the DNA was polymerized in the presence of 15 U of DNA polymerase Klenow (3'-5'exo-) and 0.2 mM dATP in a 50 μl reaction system at 37 ° C for 30 min to add A to the end. Then, the methylated adapter sequences acactctttccctacacgacgctcttccgatct (SEQ ID NO.75) and gatcggaagagcacacgtctgaactccagtcac (SEQ ID NO.76) was ligated with 50 μl of the A product in the presence of 1000 U of DNA ligase at 20°C for 15 min. The ligated product was then sulfite-converted using the EZ DNA Methylation-Gold kit from ZYMO Research. TM Kit (D5006), and the converted product was eluted into 20 μl of ultrapure water.

[0056] The bisulfite-converted product was amplified by PCR using the following reaction system: 20 μl of converted DNA; 5 μl of 10X PCR buffer; 1 μl of 10 mM dNTPs; 0.8 μl of PCR polymerase; and 1 μl each of the upstream and downstream primers aatgatacggcgaccaccgagatctacacnnnnnnnnacactctttccctacacgacgctcttccgatct (SEQ ID NO. 77) and caagcagaagacggcatacgagatgtcttggcgtgactggagttcagacgtgtgctcttccgatct (SEQ ID NO. 78). A 50-μl amplification system was performed at 95°C for 1 min, followed by 10 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 40 s, followed by extension at 72°C for 5 min. The amplified products were purified, detected by QPCR and Agilent 2100 (the purification standard was: the Agilent 2100 fragment distribution was in the range of 150bp-500bp, and the library concentration was not less than 1ng / ul), and then PE150 high-throughput whole-genome methylation sequencing was performed on the Illumina sequencing platform.

[0057] After the data was downloaded, the trim galore software was used to remove adapter sequences and low-quality sequencing reads. BSMAP software was then used for alignment. During BSMAP alignment, the positions of C bases on the reference genome were used as a guide. Ts in reads corresponding to C bases in the reference genome were converted to Cs, allowing direct alignment to the reference genome. Methylation levels were then calculated, and differentially methylated regions (DMRs) were counted using the metilene software. Finally, a combination of two statistical tests (MWU-test and 2D KS-test) was used to accurately identify differentially methylated genes.

[0058] Some gene results are shown in Figure 1 The boxed area is the differentially methylated area, the solid line is the control group, and the dotted line is the simulated methylation level curve of the diseased group. The analysis results showed that the ARHGEF3, ATP7B, C14orf102, LDLRAD4, CoL2A1, HTR7, LCP2, NCAM2, and PFKP gene intervals were hypomethylated in the diseased group; and the BANP, LOC100134868, MAD1L1, MAPK1, PALM2-AKAP2, PRDM8, PRDM16, RRBP1, and SPPL2B gene intervals were hypermethylated in the diseased group.

[0059] Through further experiments and analysis, the inventors found DNA methylation markers as shown in Table 1-1 below.

[0060] Table 1-1 Composition of DNA methylation markers

[0061]

[0062]

[0063]

[0064] Example 2 Preparation and Detection of DNA Methylation Marker Detection Kit

[0065] According to the DNA methylation markers discovered above, a detection kit is further prepared, which specifically includes a sulfite conversion reagent, a capture probe and related reagents, a library construction related reagent and a linker sequence. The probe can cover all detection sites in the marker SEQ ID NO.1-24. The basic detection process is as follows: first, the genomic DNA of the sample to be tested is treated with sulfite or bisulfite, and the cytosine (C) that is not modified by methylation in the nucleic acid is converted into uracil (U); secondly, the molecular marker sequence in a large amount of genomic DNA is obtained by fishing and amplification through the capture probe, and the conversion of uracil (U) to thymine (T) in the sequence is completed. At the same time, the amplified product of the fishing sequence is molecularly labeled to identify the source of the template; finally, the amplified product is connected to the linker under the action of nucleotide kinase and DNA ligase to construct a sequencing library.

[0066] The capture probe sequences are shown in Table 2-1 below as SEQ ID NO. 25 to SEQ ID NO. 72, and the concentration of each probe is 1 uM.

[0067] Table 2-1 Methylation marker capture probe sequences

[0068]

[0069]

[0070] Note: N12 in the probe sequences 49-72 is a tag sequence used to identify the source of the template during amplification so that the same template products can be removed during analysis to improve the accuracy of methylation detection. N is a random base.

[0071] Container 1 contains a probe mixture; a PCR reaction premix consisting of a PCR amplification enzyme, wherein the PCR enzyme is Taq enzyme, including 2X PCR buffer, 0.25 mM dNTP and 5 U PCR amplification enzyme;

[0072] Container 2 contains library construction reagents, including library construction enzymes of nucleotidyl kinase (7 U / ul) and DNA ligase (200 U / ul) and reaction buffer (200 mM Tris-HCl, 25 mM MgCl2, 15 mM DTT, 3 mM ATP, 21% PEG);

[0073] Container 3 is the library adapter sequence, specifically shown as SEQ ID NO.73 to SEQ ID NO.74 in Table 2-2 below;

[0074] Table 2-2

[0075] name SEQ ID NO. sequence Connector 1 73 aatgatacggcgaccaccgagatctacactctttccctacacgacgctcttccgatct Connector 2 74 gatcggaagagcacacgtctgaactccagtcacnnnnnnnnatctcgtatgccgtcttctgcttg

[0076] Note: N8 in the adapter sequence No. 74 is the tag sequence, which is a known sequence corresponding to different samples and is used to distinguish sample sequencing data.

[0077] The kit detection process is as follows:

[0078] Sulfite or bisulfite conversion of the genomic DNA of the sample to be tested: the conversion reagent is EZ DNAMethylation-Gold TM Kit (ZYMO Research); or refer to the literature (Quantitative Sequencing of 5-Methyl-cytosine and 5-Hydroxymethylcytosine at Single-Base Resolution.DOI:10.1126 / science.1220671, Base-Resolution Analysis of 5-Hydroxymethylcytosine in the Mammalian Genome.DOI:10.1016 / j.cell.2012.04.027, Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation.DOI:10.4161 / epi.24280). Before sulfite conversion, the samples were pretreated or replaced with potassium perruthenate / TET protein / T4 phage β-glucosyltransferase.

[0079] Molecular marker methylation information acquisition: 2 μl of probe mixture (including all probes 25-74, each probe concentration 1 μM), 25 μl of PCR reaction premix, 10-100 ng of sulfite-converted DNA template, and the total reaction volume is made up to 50 μl with ultrapure water. Amplification is performed on a PCR instrument using a gradient amplification procedure as shown in Table 2-3 below:

[0080] Table 2-3

[0081]

[0082] Amplification product library construction: 100 ng of the PCR amplification product was purified with 0.8x Beckman XP magnetic beads and rapidly constructed using the library construction enzyme and buffer system. After purification, the product was sequenced using an Agilent 2100 and QPCR quality control (the purification standard was: the Agilent 2100 fragment distribution was in the range of 150 bp-500 bp, and the library concentration was not less than 1 ng / ul). The specific library construction system is shown in Table 2-4 below:

[0083] Table 2-4

[0084] PCR amplification product (50ng) 10ul Reaction buffer 17.5ul Library construction enzyme 3ul Linker 1 / 2 (SEQ ID NO.73-SEQ ID NO.74, 10 mM) 1ul Ultrapure water 18.5ul

[0085] After mixing, the reaction was carried out at 20°C for 30 minutes. The reaction product was purified by 0.9x Beckman XP magnetic beads, and the DNA was eluted into 20ul TE eluent for quality control sequencing.

[0086] Methylation assessment of sequencing data

[0087] The data was split into different sample data using the tag (N8) in SEQ ID NO. 74. After removing low-quality sequencing data, the sequencing data was further demultiplexed using the tag (N12) in SEQ ID NO. 49-SEQ ID NO. 72 to remove duplicate amplicons generated by amplification of different markers. The methylation target molecular marker sequence was uniquely aligned with the sequencing data to calculate the methylation level.

[0088] Example 3: Detection of methylation specificity in ALL leukemia by marker amplicon sequencing

[0089] Selection of test samples: Peripheral blood samples of 141 patients with acute lymphoblastic leukemia diagnosed in Beijing 307 Hospital were selected, and genomic DNA was extracted for methylation detection. The remission group (Group A) included 109 remission cases, and the relapse group (Group B) included 32 relapse cases.

[0090] Genomic DNA extraction: Tiangen Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304-03) was used to extract genomic DNA from the samples to be tested, and the DNA concentration was quantified using the Qubit fluorescence method.

[0091] Genomic DNA sulfite conversion: Take 500ng of extracted genomic DNA in a volume of no more than 20ul and use ZYMOResearch's EZ DNAMethylation-Gold TM Kit (D5006) sulfite conversion kit, according to the kit's operating instructions, the whole genomic DNA was treated with sulfite and purified, and the DNA was eluted into 10ul EB buffer.

[0092] The probe mixture in container 1 (including all probes 25-74, each probe concentration 1uM) and PCR enzyme reaction solution are used to amplify the methylation information of all CG sites in the genomic marker sequence. The reaction system is configured as follows:

[0093] 5ul of sulfite-converted genomic DNA, 2ul of probe mix, 25ul of 2X PCRReady Mix, and 18ul of ultrapure water were added. Amplification was then performed on a PCR instrument according to the protocol shown in Table 3-1. The amplified product was purified and its concentration was determined using a Qubit assay.

[0094] Table 3-1

[0095]

[0096] By combining the enzyme preparation in container 2 and the adapter sequence in container 3, a sequencing library is quickly constructed. The reaction system is shown in Table 3-2:

[0097] Table 3-2

[0098] PCR amplification product (50ng) 10ul Reaction buffer 17.5ul Library construction enzyme 3ul Linker 1 / 2 (SEQ ID NO.73-SEQ ID NO.74, 10 mM) 1ul Ultrapure water 18.5ul

[0099] After mixing, the reaction was carried out at 20°C for 30 minutes. The reaction product was purified by 0.9x Beckman XP magnetic beads, and the DNA was eluted into 20ul eluate for quality control sequencing.

[0100] Methylation assessment of sequencing data

[0101] The data was split into different sample data using the tag (N8) in the SEQ ID NO.74 sequence. After removing the low-quality sequencing data, the repeated amplicon sequencing data formed by amplification of different markers was further removed using the tag (N12) in the SEQ ID NO.49-SEQ ID NO.72 sequence. The methylation target molecular marker sequence was uniquely aligned with the sequencing data to calculate the methylation level, such as Figure 2The heatmap results showed that the methylation levels of each marker in the relapse group B and the remission group A were exactly the same as the whole gene methylation trends, that is, the ARHGEF3, ATP7B, C14orf102, LDLRAD4, CoL2A1, HTR7, LCP2, NCAM2, and PFKP gene intervals were hypomethylated in the relapse group B; the BANP, LOC100134868, MAD1L1, MAPK1, PALM2-AKAP2, PRDM8, PRDM16, RRBP1, and SPPL2B gene intervals were hypermethylated in the relapse group B. Further principal component analysis was performed on the methylation data of the 24 markers detected in the two groups of patients, and the two most important dimensions PC1 and PC2 were selected for dimensionality reduction analysis. The analysis results are as follows: Figure 3 As shown in the figure, the methylation levels of the two groups of patients were effectively separated, and the remission and relapse patients were clearly distinguished by the corresponding methylation data.

[0102] The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0103] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and these variations and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims. Sequence Listing <110> high flying Xi Yongzhi <120> DNA methylation markers in acute lymphoblastic leukemia and their applications <160> 78 <170> SIPOSequenceListing 1.0 <210> 1 <211> 80 <212> DNA <213> Artificial Sequence <400> 1 cgaggcaccc agaggtagaa aagatctcct ggtcacagcc acggggtcca gaggacagtc 60 tctgtgactt tggggttccg 80 <210> 2 <211> 512 <212> DNA <213> Artificial Sequence <400> 2 cgcaccccgt gggcacttcg gccggcagat gccttctcct cctgttcaga tccctggaat 60 agttccaagc aactgcagtc agtgccatca gaggcgcctc aaactgtacc gaagacccta 120 gcaaggcgag ttggaggaaa catgagtcaa aaccaacaca gctgccagcg gcttggctcc 180 ctgtctgaac gttttcactt ctcagaaact ttcatgcaat taatttataa gagaagaaaa 240 aaaaaagaag ccaaggcatg tgactgctgc agacccatgg cacagcagag cccagcatgc 300 caggtgccgt cacacagggt cctgtttggg ggcagcgcag cacagtggct gaggggggct 360 ctgtgcctcc agggttccaa tcccagctct gctgcctgcc tgccatggac tctggggata 420 ttactccacc tcactgcact tggcttttcc tgaggaaagc tgggatgatg acagtgctgc 480 ctcacggaac agctacagag tggaactaga cg 512 <210> 3 <211> 67 <212> DNA <213> Artificial Sequence <400> 3 cgtgacgatc acctgacgct tgctgtgtga tgggggcagc tggtctcagc tgtgcctggg 60 agggtcg 67 <210> 4 <211> 41 <212> DNA <213> Artificial Sequence <400> 4 cgatccgccc gcctcagcac tttgggaggc caaggtgggc g 41 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <400> 5 cggaccgtga gaagacacag gtgccccg 28 <210> 6 <211> 420 <212> DNA <213> Artificial Sequence <400> 6 cggagggcac agggagcagg ggtttcagcg ccacactgtt cattcccact gcggctggag 60 cgctggctcg ctgtgccctg ctgctctcac tcctgcccac ctcatacgtt tagaaaagca 120 tgtggaaaac gatggaaagg aatgattttc agtaaggctt gaaggagtgg actaaagggc 180 ttactgatgc agttgtactc ggtctgcctc tgacactcca gactaaagaa aagagccaca 240 ggcttcctct ggcatcattg tggggatgaa ccgcagggga ccgtttccct gcaggcaagg 300 cagaccctgc tgcaccacag accagatgtg tgtgtttgtc ggttgtccag tgacagctct 360 ccgttactgc atttcacgga aactgcattt ccaagagctt agggtccggc gggttggccg 420 <210> 7 <211> 334 <212> DNA <213> Artificial Sequence <400> 7 cgcgggccaa ccctcagccc tgctccaggc ggtttgggca caggcagctc ttctctctgg 60 cagccccact caccgtgcag ccatccttca gggcagtgta cgtgaacctg ctattgccct 120 ctgcccggat ctccacgtca ttggagccct ggatgagcag ggccttcttg aggttgccag 180 ctgcttcgtc cagataggca atgctgttct tgcagtggta ggtgatgttc tgggagcctt 240 ccgtggacag caggcgtagg aaggtcatct ggacgttggc agtgttggga gccagattgt 300 catctccata gctgaactgt tggggcagag agcg 334 <210> 8 <211> 111 <212> DNA <213> Artificial Sequence <400> 8 cgctttcttt ggtttgttat ctgtggtgaa ccacggtccg aaaatattaa atggaaattt 60 tcagaaataa acaatgtgta agttgtcaat tgcacactat tctgagtagc g 111 <210> 9 <211> 142 <212> DNA <213> Artificial Sequence <400> 9 cgagcagcag cctcatatcc aagaactgaa agtcattttg ttgacagaca aaaagggatt 60 gaggccacag aaagctaaat gccttgctca gagcatagcc aagaaagggg tgggggcagc 120 tgggattctg ggctcctttc cg 142 <210> 10 <211> 34 <212> DNA <213> Artificial Sequence <400> 10 cggaggattc tgcaggccaa taccacgtct cgcg 34 <210> 11 <211> 438 <212> DNA <213> Artificial Sequence <400> 11 cggctaaaga gctgccccac ttgcctgcag gctgaccccg ggccctctcc agcgccccca 60 ggacaggcac tccctggcga agcgcaggtg tgggctgcag ccctggcacc gcaaagtagg 120 gctcagagaa gggagtgtgg agctgcgagg caacaactca gtaaccacaa tgggcagaag 180 acaggccatc agcaaaccag caagcaggtg cctcctgaca ctggagggga aaccggaggc 240 tgggggggcc ctcggggagg ggtggcggga cagcctgtgg tgtcagagac aggcggtgtc 300 agagccgggg acgcttccaa tgggcagctg gcgtgctcat tcgtggtcag cccagtgggc 360 ccgaggttgg gccaaccggg agcaatgggc aaggtcacaa gaggacgagc aaaccaggaa 420 cggcctctgc acgggacg 438 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <400> 12 cgcaaacaaa aacactggca ctacttaatg aagcg 35 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <400> 13 cgccactgta ctccagcctg ggcg 24 <210> 14 <211> 262 <212> DNA <213> Artificial Sequence <400> 14 cggtcaggca cggtggctca cgcctgtaat cccaacactt tgggaggcca aggtgggcag 60 atcacaaggt caggagatcg agaccatcct ggctatcatg gtgaaacccc gtctctacta 120 aaaatacaaa aaattagcca ggggtggtgg cgagcgcctg tagtcccagc tacttgggag 180 gctgaggcag gagaacagtg tgaacccggg aggcggagct tgcagtgagc cgagatcacg 240 ccactgtact ccagcctggg cg 262 <210> 15 <211> 165 <212> DNA <213> Artificial Sequence <400> 15 cgtgagggtt ctcagaagtc gcatgactca tatagtccgt caactctcag gaatgggttc 60 tgagaattcc aagtaagaca accccatgcc tgaaaaaaaa aaaaaaaagc cttgaaatct 120 ttattgtatt tcattaacag ttttactagc tgttactttt caacg 165 <210> 16 <211> 47 <212> DNA <213> Artificial Sequence <400> 16 cgaactgagg agggagttag ctttctccaa ctctgggcag attaccg 47 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <400> 17 cgtagagcaa cagatacg 18 <210> 18 <211> 104 <212> DNA <213> Artificial Sequence <400> 18 cgaataagtt gaaatgactt cagagcctgt ggcatgttct tagatgtgat cgaatgactc 60 aacgtaaaaa attctgtccg acgatggatc cgatacagcc aacg 104 <210> 19 <211> 61 <212> DNA <213> Artificial Sequence <400> 19 cgtggccaag tgaagaagag tcgcctgcac ttacaaaggg cctctgcagc ctggcgtgac 60 g 61 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <400> 20 cgtgctgccc ccacctgcg 19 <210> 21 <211> 62 <212> DNA <213> Artificial Sequence <400> 21 cggcgccgga gggagcgcct ggcccagcaa agagttaaag ggaggggacg tgggctgtca 60 cg 62 <210> 22 <211> 31 <212> DNA <213> Artificial Sequence <400> 22 cgagactcac aggacaagaa cgatgcctcc g 31 <210> 23 <211> 52 <212> DNA <213> Artificial Sequence <400> 23 cgcctgtccc tggagggggg cagtgcttcg cggcagtgga aatttgagtc cg 52 <210> 24 <211> 305 <212> DNA <213> Artificial Sequence <400> 24 cgattgtggg gaccggggct ccgtgggctt tcaccatgct gccaccccga ggtcaccagg 60 gccttgaccc agatgctccc agaccaggcc ggagactttg catgatttca gtgatgaggg 120 cccggctgtt ggagagcccg tctgtgcctg tgtgctgggg ttggtgtctg gcaccaggga 180 cctttgggac ctttagccaa gccagagctg tccttcccca gtcctagctt ggcagagctg 240 cgtctgcact gttttccctt gtggcgttgt ggtgtcggcc cgtttggtgc gcagctcagc 300 atccg 305 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <400> 25 ggtttttatg ggaaggttag ttatg 25 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <400> 26 gaattgagtt atgataggtg ttgta <210> 27 <211> 30 <212> DNA <213> Artificial Sequence <400> 27 ttatataat gtttttttaa atgaaggtat <210> 28 <211> 26 <212> DNA <213> Artificial Sequence <400> 28 ttgttatttt total total <210> 29 <211> 25 <212> DNA <213> Artificial Sequence <400> 29 ttgtgtttt atttttttga tgttt <210> 30 <211> 28 <212> DNA <213> Artificial Sequence <400> 30 gagttgtttt atttgtttgt aggttgat <210> 31 <211> 30 <212> DNA <213> Artificial Sequence <400> 31 father gtagttagta taaagggtgt <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <400> 32 ttagtttggg ttagtagtt aaatt <210> 33 <211> 30 <212> DNA <213> Artificial Sequence <400> 33 agaagagttt gaaagaata attack <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <400> 34 gggttttgga ttatttttt <210> 35 <211> 30 <212> DNA <213> Artificial Sequence <400> 35 tttttttgtt gtgattaata ttttagaggt <210> 36 <211> 29 <212> DNA <213> Artificial Sequence <400> 36 tttaatgtgt gagtatgtag 29. tttaatgtgt <210> 37 <211> 25 <212> DNA <213> Artificial Sequence <400> 37 agtattgta tttggtgttg gaggt <210> 38 <211> 25 <212> DNA <213> Artificial Sequence <400> 38 ttataggtag tgttttggga ttttt 25 <210> 39 <211> 25 <212> DNA <213> Artificial Sequence <400> 39 aaatgttttt gtttttgttt ttttt 25 <210> 40 <211> 25 <212> DNA <213> Artificial Sequence <400> 40 atatggtttg gttttttttg tattg 25 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <400> 41 ttggtaagtg gttagaggaa atagaat 27 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <400> 42 ggatttatgt aataagtatt ttgaaagaaa 30 <210> 43 <211> 26 <212> DNA <213> Artificial Sequence <400> 43 ttttagggtt agagaatttt tggagt 26 <210> 44 <211> 26 <212> DNA <213> Artificial Sequence <400> 44 gtttattttt aattttgttt tttata 26 <210> 45 <211> 30 <212> DNA <213> Artificial Sequence <400> 45 tgggtagtat gattagttta gatagtttta 30 <210> 46 <211> 26 <212> DNA <213> Artificial Sequence <400> 46 ttgtgtttgt ttatttttta ggatgg 26 <210> 47 <211> 25 <212> DNA <213> Artificial Sequence <400> 47 tttagtttgg atgataggtt gtttg 25 <210> 48 <211> 27 <212> DNA <213> Artificial Sequence <400> 48 ggtagttgtg ggaaggaagt agtatta 27 <210> 49 <211> 39 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 49 nnnnnnnnnn nnaaactcta aaaaaaccca aaaaatatc 39 <210> 50 <211> 37 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 50 nnnnnnnnnn nnactttcct caaaaaaaac caaatac 37 <210> 51 <211> 42 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 51 nnnnnnnnnn nnaaaaacta ctaaaaaatt ttaaaactac aa 42 <210> 52 <211> 37 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 52 nnnnnnnnnn nnaactaact ccctactacc ccaatac 37 <210> 53 <211> 42 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 53 nnnnnnnnnn nnattaaata atttacctaa atttacctaa at 42 <210> 54 <211> 37 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 54 nnnnnnnnnn nntactcctt accttcctac ccctcta 37 <210> 55 <211> 39 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 55 nnnnnnnnnn nncctaataa aaaataatca ttactaacc 39 <210> 56 <211> 36 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 56 nnnnnnnnnn nntaatcacc aaaatcccac aataac 36 <210> 57 <211> 42 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 57 nnnnnnnnnn nntatccatt taaacataaa taaaatataa at 42 <210> 58 <211> 35 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 58 nnnnnnnnnn nncacctaca caaccacaaa cctac 35 <210> 59 <211> 35 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 59 nnnnnnnnnn nnctcccaaa atccccaata aatac 35 <210> 60 <211> 42 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 60 nnnnnnnnnn nnatcaatct taacacacta aataacaaaa cc 42 <210> 61 <211> 38 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 61 nnnnnnnnnn nnctcaaaaa aaatcaaaac atttatcc 38 <210> 62 <211> 39 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 62 nnnnnnnnnn nnccttactc actcaaacaa acatactac 39 <210> 63 <211> 42 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 63 nnnnnnnnnn nnttttatct aaccacctat taatttctat ct 42 <210> 64 <211> 42 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 64 nnnnnnnnnn nnaaaactca catattttct tcttctaact aa 42 <210> 65 <211> 42 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 65 nnnnnnnnnn nnttcaatct taaaacttca aaataaaaaa ta 42 <210> 66 <211> 40 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 66 nnnnnnnnnn nnaaaaaaaa caaaacataa aacctttaac 40 <210> 67 <211> 35 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 67 nnnnnnnnnn nnaaaaaaat tatacaaata aaccc 35 <210> 68 <211> 42 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 68 nnnnnnnnnn nnaaccatac ctaataattc tttctactaa at 42 <210> 69 <211> 37 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 69 nnnnnnnnnn nnaaacttaa tcacaaaaac taaaccc 37 <210> 70 <211> 37 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 70 nnnnnnnnnn nnaaaaaaac tatctcccac tacaacc 37 <210> 71 <211> 38 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 71 nnnnnnnnnn nncccttcta aaaaaactaa aaaattcc 38 <210> 72 <211> 37 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(12) <223> n is a, c, g, or t <400> 72 nnnnnnnnnn nnctataaac ctcccaaaca aaaaaac 37 <210> 73 <211> 58 <212> DNA <213> Artificial Sequence <400> 73 aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58 <210> 74 <211> 65 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (34)..(41) <223> n is a, c, g, or t <400> 74 gatcggaaga gcacacgtct gaactccagt cacnnnnnnn natctcgtat gccgtcttct 60 gcttg 65 <210> 75 <211> 33 <212> DNA <213> Artificial Sequence <400> 75 acactctttc cctacacgac gctcttccga tct 33 <210> 76 <211> 33 <212> DNA <213> Artificial Sequence <400> 76 gatcggaaga gcacacgtct gaactccagt cac 33 <210> 77 <211> 70 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (30)..(37) <223> n is a, c, g, or t <400> 77 aatgatacgg cgaccaccga gatctacacn nnnnnnnaca ctctttccct acacgacgct 60 cttccgatct 70 <210> 78 <211> 66 <212> DNA <213> Artificial Sequence <400> 78 caagcagaag acggcatacg agatgtcttg gcgtgactgg agttcagacg tgtgctcttc 60 cgatct 66

Claims

1. A DNA methylation marker combination for detecting acute lymphoblastic leukemia, characterized in that: The DNA methylation marker combination consists of genes ARHGEF3, ATP7B, C14orf102, CoL2A1, LCP2, MAD1L1, MAPK1, LOC100134868, HTR7, BANP, LDLRAD4, NCAM2, PALM2-AKAP2, RRBP1 and SPPL2B, PFKP, PRDM8, and PRDM16; the sequence of ARHGEF3 is shown in SEQ ID NO.1 or its complementary sequence, the sequence of ATP7B is shown in SEQ ID NO.2 or its complementary sequence, the sequence of C14orf102 is shown in SEQ ID NO.4 or its complementary sequence, the sequence of LDLRAD4 is shown in SEQ ID NO.5-SEQ ID NO.6 or the complementary sequence of SEQ ID NO.5-SEQ ID NO.6, the sequence of CoL2A1 is shown in SEQ ID NO.7, the sequence of HTR7 is shown in SEQ ID NO.8, and the sequence of LCP2 is shown in SEQ ID NO. No. 9 or its complementary sequence, the sequence of NCAM2 is shown in SEQ ID No. 15 or its complementary sequence, the sequence of BANP is shown in SEQ ID No. 3 or its complementary sequence, the sequence of LOC100134868 is shown in SEQ ID No. 10 or its complementary sequence, the sequence of MAD1L1 is shown in SEQ ID No. 11 or its complementary sequence, the sequence of MAPK1 is shown in SEQ ID No. 12-SEQ ID No. 14 or the complementary sequence of SEQ ID No. 12-SEQ ID No. 14, the sequence of PALM2-AKAP2 is shown in SEQ ID No. 16-SEQ ID No. 17 or the complementary sequence of SEQ ID No. 16-SEQ ID No. 17, the sequence of RRBP1 is shown in SEQ ID No. 22 or its complementary sequence, the sequence of SPPL2B is shown in SEQ ID No. 23-SEQ ID No. 24 or the complementary sequence of SEQ ID No. 23-SEQ ID No. 24; the sequence of PFKP is shown in SEQ ID No. The sequence of PRDM16 is shown in SEQ ID NO.19-SEQ ID NO.20 or the complementary sequence of SEQ ID NO.19-SEQ ID NO.20; the sequence of PRDM8 is shown in SEQ ID NO.21 or its complementary sequence.

2. The DNA methylation marker combination according to claim 1, characterized in that The DNA methylation marker combination includes sequences shown in SEQ ID NO.1 to SEQ ID NO.

24.

3. Use of the DNA methylation marker combination according to any one of claims 1 to 2 in the preparation of a kit for detecting, classifying or predicting, monitoring treatment, or prognosing acute lymphoblastic leukemia.

4. An acute lymphoblastic leukemia detection kit, characterized in that: Comprising a reagent for detecting the methylation degree of the DNA methylation marker combination according to any one of claims 1-2.

5. The acute lymphoblastic leukemia detection kit according to claim 4, characterized in that: The detection kit includes a capture probe for a DNA methylation marker, and the capture probe includes: For SEQ ID NO. 1, SEQ ID NO. 25 and SEQ ID NO. 49, For SEQ ID NO.26 and SEQ ID NO.50 of SEQ ID NO.2, For SEQ ID NO.36 and SEQ ID NO.60 of SEQ ID NO.3, SEQ ID NO.27 and SEQ ID NO.51 for SEQ ID NO.4, SEQ ID NO.37 and SEQ ID NO.61 for SEQ ID NO.5, SEQ ID NO.38 and SEQ ID NO.62 for SEQ ID NO.6, SEQ ID NO.28 and SEQ ID NO.52 for SEQ ID NO.7, For SEQ ID NO. 8, SEQ ID NO. 35 and SEQ ID NO. 59, SEQ ID NO.29 and SEQ ID NO.53 for SEQ ID NO.9, For SEQ ID NO. 10, SEQ ID NO. 34 and SEQ ID NO. 58, For SEQ ID NO. 11, SEQ ID NO. 30 and SEQ ID NO. 54, For SEQ ID NO.12, SEQ ID NO.31 and SEQ ID NO.55, For SEQ ID NO.13, SEQ ID NO.32 and SEQ ID NO.56, For SEQ ID NO.14, SEQ ID NO.33 and SEQ ID NO.57, For SEQ ID NO.15, SEQ ID NO.39 and SEQ ID NO.63, SEQ ID NO.40 and SEQ ID NO.64 for SEQ ID NO.16, For SEQ ID NO. 17, SEQ ID NO. 41 and SEQ ID NO. 65, SEQ ID NO.46 and SEQ ID NO.70 for SEQ ID NO.22, SEQ ID NO. 47 and SEQ ID NO. 71 for SEQ ID NO. 23, and SEQ ID NO. 48 and SEQ ID NO. 72 for SEQ ID NO. 24.

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