A combination of DNA methylation markers, a reagent kit, an apparatus, and a storage medium

Abstract

This invention provides a DNA methylation marker combination, kit, device, and storage medium, belonging to the field of biomedical diagnostics. It includes methylation sites in target regions of the FGGY, LMNA, REST, PLOD2, POLR1D, NFKBIZ, PRKCB, CCSER1, and NEFM genes. The target regions of the FGGY and LMNA genes are selected from Chr1, the target regions of the REST and CCSER1 genes are selected from Chr4, the target regions of the PLOD2 and NFKBIZ genes are selected from Chr3, the target region of the POLR1D gene is selected from Chr13, the target region of the PRKCB gene is selected from Chr16, and the target region of the NEFM gene is selected from Chr8. This invention is applicable to adult diffuse gliomas and offers advantages such as simplicity, economy, speed, and clinical applicability.

Description

Technical Field

[0001] This invention relates to the field of biomedical diagnostics, specifically to a combination of DNA methylation markers, a reagent kit, an apparatus, and a storage medium. Background Technology

[0002] Diffuse gliomas in adults are among the most common, complex, and malignant tumors of the central nervous system, including astrocytomas, oligodendrogliomas, and glioblastomas. The median survival for oligodendrogliomas is 60–119 months, for astrocytomas it is 18–36 months, and for glioblastomas, which are even more malignant, the median survival is only 8 months. Accurate diagnosis of diffuse gliomas in adults is crucial for surgical and postoperative treatment.

[0003] According to the fifth edition of the Central Nervous System (CNS) Tumor Classification published by the World Health Organization (WHO) in 2021, the molecular subtyping diagnostic criteria for adult diffuse gliomas are as follows: (1) Astrocytoma, IDH-mutant type, without 1p19q co-deletion (A); (2) Oligodendroglioma, IDH-mutant type and 1p / 19q co-deletion (O); (3) Glioblastoma, IDH wild-type (GBM): IDH wild-type, H3 wild-type, and having one or more of the following characteristics: TERT promoter mutation, +7 / -10, EGFR amplification (EGFRamp), microvascular proliferation, necrosis. Therefore, in current clinical practice, the molecular subtyping of adult diffuse gliomas relies on multiple technologies or multiple platforms for detection. For example, IDH, H3, and TERT mutations require Sanger sequencing or next-generation sequencing, while 1p19q co-deletion, EGFR amplification, and +7 / -10 require fluorescence in situ hybridization (FISH) or copy number variation analysis. However, this method, which relies on multiple technologies or multiple platforms for detection, has limitations in clinical practice: (1) it is difficult to complete all molecular detection when the amount of tissue sample is limited; (2) the results of different detection platforms may be inconsistent (such as the difference in interpretation of 1p19q co-deletion between FISH and NGS technologies); (3) it is time-consuming due to its reliance on multiple detection platforms.

[0004] DNA methylation is a common epigenetic modification that is closely related to the occurrence and development of cancer. Numerous studies have shown that DNA methylation is associated with disease typing, malignancy, and recurrence. In the field of central nervous system tumors, studies have shown that DNA methylation can be used to assist in the typing of CNS tumors (MNP; https: / / www.molecularneuropathology.org / mnp / ). This classifier is based on Illumina's 450k, 850k, and 935k chips. Methylation was detected in more than 2,800 samples. Through PCA analysis, 91 subclasses were obtained. The machine learning method of random forest was used to screen important typing probes and construct a typing model to provide typing guidance for CNS tumors. Although the molecular typing method based on DNA methylation is based on a single detection platform, it is more convenient than multi-technology and multi-platform detection such as DNA mutation, gene amplification, and chromosome copy number variation. However, this classifier has significant limitations in clinical application: (1) high cost and high technical requirements. (1) It relies on Illumina methylation chips, but the sample detection cost is high, and it requires professional experimental procedures and instrument platforms, which places extremely high demands on the hardware and personnel of medical institutions. (2) The time cycle is long. It takes about 2 weeks from experimentation to genotyping analysis for methylation chip detection technology. (3) The classifier is highly dependent on the reference dataset and model built in advance by overseas research teams, and cannot be implemented and used in Chinese hospitals. (4) Accessibility and compliance risks: At present, the website has suspended new user registration and the server is located overseas. Data upload involves cross-border transmission risks of human genetic resource information. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a DNA methylation marker combination, a reagent kit, an apparatus, and a storage medium. The technical solution provided by this invention utilizes qPCR and is effective for adult diffuse gliomas, offering advantages such as simplicity, economy, speed, and clinical applicability.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a combination of DNA methylation markers for the subtyping of adult diffuse gliomas for non-disease diagnosis and treatment purposes, comprising: target regions of the FGGY, LMNA, REST, PLOD2, POLR1D, NFKBIZ, PRKCB, CCSER1, and NEFM genes; using hg19 as a reference genome, the target region of the FGGY gene is selected from Chr1: 59762781-59762859, the target region of the LMNA gene is selected from Chr1: 156085007-156085095, the target region of the REST gene is selected from Chr4: 57773841-57773938, and the target region of the NFKBIZ, PRKCB, CCSER1, and NEFM genes... The target region of the LOD2 gene is selected from Chr3: 145879404-145879505, the target region of the POLR1D gene is selected from Chr13: 28196336-28196420, the target region of the NFKBIZ gene is selected from Chr3: 101569020-101569114, the target region of the PRKCB gene is selected from Chr16: 23847529-23847601, the target region of the CCSER1 gene is selected from Chr4: 91048392-91048494, and the target region of the NEFM gene is selected from Chr8: 24772267-24772341.

[0008] This invention also provides a kit comprising: an upstream primer and a downstream primer, wherein the upstream primer and the downstream primer are used to detect the above-mentioned nine methylation markers; the upstream primer is shown in SEQ ID No: 1-9; and the downstream primer is shown in SEQ ID No: 10-18.

[0009] Furthermore, it also includes probes corresponding to the upstream and downstream primers, as shown in SEQ ID No: 19-27.

[0010] Furthermore, it also includes an internal reference gene, which is the GAPDH gene. The primers for the GAPDH gene are shown in SEQ ID No: 28-29, and the probe for the GAPDH gene is shown in SEQ ID No: 30.

[0011] This invention also provides an apparatus for typing adult diffuse gliomas, comprising: a detection unit, wherein the detection unit uses the above-mentioned kit to detect the methylation level of the target region of the gene and obtains a detection result; and an analysis unit, wherein the analysis unit determines the typing of adult diffuse gliomas based on the detection result.

[0012] Furthermore, the detection unit includes a real-time PCR instrument.

[0013] Furthermore, the analysis unit determines the adult diffuse glioma subtype based on the ΔCt value of each gene; wherein the ΔCt value is the corresponding gene Ct value minus the internal reference gene Ct value.

[0014] Furthermore, if the ΔCt of the gene is not greater than a preset value, it is determined to be positive; otherwise, it is determined to be negative.

[0015] Furthermore, the methylation detection results for each gene target region are represented by a score S, with a positive value of 1 and a negative value of 0. Adult diffuse glioma classification rules: 1) First, analyze the methylation detection results of the POLR1D and NFKBIZ gene target regions, MEscore1=S POLR1D +S NFKBIZ When MEscore1=2, the sample is identified as IDH mutant; otherwise, the sample is identified as glioblastoma, IDH wild-type. 2) When the sample is identified as IDH mutant, the methylation detection results of the target regions of the REST, PLOD2, FGGY, and LMNA genes are analyzed, and MEscore2=S REST +S PLOD2 - S FGGY -S LMNA When MEscore2≥1, the sample is identified as oligodendroglioma, with IDH mutation and 1p19q co-deletion; otherwise, the sample is identified as astrocytoma, with IDH mutation and no 1p19q co-deletion; 3) When the sample is identified as glioblastoma, with IDH wild-type, the methylation detection results of the target regions of PRKCB, CCSER1, and NEFM genes are analyzed, and MEscore3=S PRKCB + S CCSER1 + S NEFM When MEscore3≥2, the sample is identified as glioblastoma carrying molecular characteristics, IDH wild type; otherwise, the sample is identified as glioblastoma with only pathological characteristics, IDH wild type.

[0016] This invention also provides a computer-readable storage medium storing one or more programs that can be executed by one or more processors to implement the following method: analyzing the results detected by the above-described kit; calculating the ΔCt of the corresponding gene, determining a positive result if the ΔCt of the gene is not greater than a preset value, and a negative result otherwise; representing the methylation detection result of the target region of each gene with a score S, where a positive result is 1 and a negative result is 0; MEscore1=S POLR1D +S NFKBIZWhen MEscore1=2, the sample is identified as IDH mutant; otherwise, it is identified as IDH wild-type, glioblastoma. When the sample is identified as IDH mutant, methylation detection results of the target regions of the REST, PLOD2, and LMNA genes are analyzed. When MEscore2=S REST +S PLOD2 - S FGGY -S LMNA When MEscore2 ≥ 1, the sample is classified as oligodendroglioma, with both IDH mutation and 1p19q co-deletion; otherwise, the sample is classified as astrocytoma, with no 1p19q co-deletion in the IDH mutation. When the sample is classified as IDH wild-type glioblastoma, methylation detection results of the target regions of PRKCB, CCSER1, and NEFM genes are analyzed, and MEscore3 = S PRKCB + S CCSER1 +S NEFM When MEscore3≥2, the sample is identified as glioblastoma carrying molecular characteristics, IDH wild type; otherwise, the sample is identified as glioblastoma with only pathological characteristics, IDH wild type.

[0017] The beneficial effects of the technical solutions provided by the embodiments of the present invention include:

[0018] The technical solution provided by this invention employs a single-atom detection method, requiring only the detection of nine methylation markers for genotyping, which is simpler than the guideline-recommended method of mutation combined with CNV detection (IDH1, IDH2, H3, 1p19q, TERT228, TERT250, EGFRamp, +7 / -10). Secondly, the detection cost of qPCR used in this invention is lower than that of 450k chip detection, and the operation is simple, the results are easy to interpret, and it does not depend on the chip's reference dataset and reference model. Finally, the technical solution of this invention has strong clinical applicability. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The classification rules for adult diffuse gliomas based on nine methylation markers provided in this embodiment of the invention;

[0021] Figure 2 The results of retrospective cohort methylation marker detection provided in Example 2 of this invention;

[0022] Figure 3 The results of prospective cohort methylation marker detection provided in Embodiment 3 of the present invention. Detailed Implementation

[0023] The present invention will be further described in detail below through specific embodiments. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be obtained commercially.

[0024] As used herein, the terms “comprising,” “including,” “having,” or any other variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, article, or apparatus that includes listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Unless the context clearly specifies otherwise, the singular forms “an” and “the” include a plural of objects under discussion.

[0025] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, this invention can be implemented using any prior art methods, apparatus, and materials similar to or equivalent to those described in the embodiments of this invention, based on the knowledge of those skilled in the art and the description of this invention.

[0026] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, immunology, laboratory medicine, gene sequencing technology, bioinformatics technology, and related fields.

[0027] This invention provides a DNA methylation biomarker combination for adult diffuse glioma subtyping for non-disease diagnosis and treatment purposes, comprising: target regions of the FGGY, LMNA, REST, PLOD2, POLR1D, NFKBIZ, PRKCB, CCSER1, and NEFM genes; using hg19 as a reference genome, the target region of the FGGY gene is selected from Chr1: 59762781-59762859, the target region of the LMNA gene is selected from Chr1: 156085007-156085095, and the target region of the REST gene is selected from Chr4: 57773841-57773938. The target region of the PLOD2 gene is selected from Chr3: 145879404-145879505, the target region of the POLR1D gene is selected from Chr13: 28196336-28196420, the target region of the NFKBIZ gene is selected from Chr3: 101569020-101569114, the target region of the PRKCB gene is selected from Chr16: 23847529-23847601, the target region of the CCSER1 gene is selected from Chr4: 91048392-91048494, and the target region of the NEFM gene is selected from Chr8: 24772267-24772341.

[0028] The technical solution provided by this invention uses a single mic detection method, which only needs to detect 9 methylation markers for genotyping, making it simpler than the guideline-recommended method of mutation combined with CNV detection (IDH1, IDH2, H3, 1p19q, TERT228, TERT250, EGFRamp, +7 / -10). Secondly, the detection cost of qPCR used in this invention is lower than that of 450k chip detection, and the operation is simple and the results are easy to interpret, without relying on the chip's reference dataset and reference model. Finally, it has strong clinical applicability.

[0029] This invention also provides a kit comprising: upstream primers and downstream primers, wherein the upstream primers and downstream primers are used to detect the above-mentioned nine methylation markers; the upstream primers include: a first upstream primer as shown in SEQ ID No: 1, a second upstream primer as shown in SEQ ID No: 2, a third upstream primer as shown in SEQ ID No: 3, a fourth upstream primer as shown in SEQ ID No: 4, a fifth upstream primer as shown in SEQ ID No: 5, a sixth upstream primer as shown in SEQ ID No: 6, a seventh upstream primer as shown in SEQ ID No: 7, an eighth upstream primer as shown in SEQ ID No: 8, and a ninth upstream primer as shown in SEQ ID No: 9; the downstream primers include: a first downstream primer as shown in SEQ ID No: 10 corresponding to the first upstream primer, a second downstream primer as shown in SEQ ID No: 11 corresponding to the second upstream primer, a third downstream primer as shown in SEQ ID No: 12 corresponding to the third upstream primer, a fourth downstream primer as shown in SEQ ID No: 13 corresponding to the fourth upstream primer, and a downstream primer as shown in SEQ ID No: 9 corresponding to the fifth upstream primer. The fifth downstream primer shown in No. 14, the sixth downstream primer corresponding to the sixth upstream primer as shown in SEQ ID No. 15, the seventh downstream primer corresponding to the seventh upstream primer as shown in SEQ ID No. 16, the eighth downstream primer corresponding to the eighth upstream primer as shown in SEQ ID No. 17, and the ninth downstream primer corresponding to the ninth upstream primer as shown in SEQ ID No. 18.

[0030] Specifically, it also includes: a first probe corresponding to the first upstream primer and the first downstream primer, as shown in SEQ ID No: 19; a second probe corresponding to the second upstream primer and the second downstream primer, as shown in SEQ ID No: 20; a third probe corresponding to the third upstream primer and the third downstream primer, as shown in SEQ ID No: 21; a fourth probe corresponding to the fourth upstream primer and the fourth downstream primer, as shown in SEQ ID No: 22; a fifth probe corresponding to the fifth upstream primer and the fifth downstream primer, as shown in SEQ ID No: 23; a sixth probe corresponding to the sixth upstream primer and the sixth downstream primer, as shown in SEQ ID No: 24; a seventh probe corresponding to the seventh upstream primer and the seventh downstream primer, as shown in SEQ ID No: 25; an eighth probe corresponding to the eighth upstream primer and the eighth downstream primer, as shown in SEQ ID No: 26; and a ninth probe corresponding to the ninth upstream primer and the ninth downstream primer, as shown in SEQ ID No: 27.

[0031] Specifically, it also includes an internal reference gene, which is the GAPDH gene. The upstream primer of the GAPDH gene is shown in SEQ ID No: 28, the downstream primer of the GAPDH gene is shown in SEQ ID No: 29, and the probe of the GAPDH gene is shown in SEQ ID No: 30. The sequences of the primers and probes are shown in Table 1.

[0032] Table 1 Primer and probe sequences

[0033] This invention also provides an apparatus for typing adult diffuse gliomas, comprising: a detection unit, wherein the detection unit uses the above-mentioned kit to detect the methylation level of the target region of the gene and obtains a detection result; and an analysis unit, wherein the analysis unit determines the typing of adult diffuse gliomas based on the detection result.

[0034] Specifically, the detection unit includes a real-time PCR instrument.

[0035] The analysis unit determines the adult diffuse glioma subtype based on the ΔCt value of each gene; where the ΔCt value is the corresponding gene Ct value minus the internal reference gene Ct value.

[0036] If the ΔCt of a certain gene is not greater than a preset value, it is considered positive; otherwise, it is considered negative. In this embodiment of the invention, the preset value is 7.

[0037] The results were interpreted, and the methylation detection results for each gene target region were represented by a fraction S, with the corresponding gene labeled in the lower right corner, such as S. FGGY The results of FGGY gene methylation detection are as follows: ΔCt≤7 is positive, with a value of 1; ΔCt>7 is negative, with a value of 0; no methylation marker detected is also negative, with a value of 0.

[0038] The classification rules for methylation markers are as follows: Figure 1 As shown:

[0039] (1) First, the methylation detection results of the target regions of the POLR1D and NFKBIZ genes were analyzed, and MEscore1=S POLR1D +S NFKBIZ When all methylation test results are positive, i.e. MEscore1=2, the sample is identified as IDH mutant; otherwise, the sample is identified as glioblastoma, IDH wild type.

[0040] (2) When the sample is identified as IDH mutant, the methylation detection results of the target regions of the REST, PLOD2, FGGY, and LMNA genes are analyzed, and the scores are calculated according to the following formula: MEscore2=S REST +S PLOD2 - S FGGY -S LMNA When MEscore2≥1, the sample is identified as oligodendroglioma, with IDH mutation and 1p19q co-deletion; otherwise, the sample is identified as astrocytoma, with IDH mutation and no 1p19q co-deletion.

[0041] (3) When the sample is identified as glioblastoma, IDH wild-type, the methylation detection results of the target regions of PRKCB, CCSER1, and NEFM genes are analyzed, and the score is calculated according to the following formula: MEscore3=S PRKCB + S CCSER1 + S NEFM When MEscore3≥2, the sample is identified as glioblastoma carrying molecular characteristics, IDH wild type; otherwise, the sample is identified as glioblastoma with only pathological characteristics, IDH wild type.

[0042] An embodiment of the present invention provides a computer-readable storage medium storing one or more programs, which can be executed by one or more processors to achieve adult diffuse glioma classification;

[0043] The DNA methylation marker combination includes the target regions of the FGGY, LMNA, REST, PLOD2, POLR1D, NFKBIZ, PRKCB, CCSER1, and NEFM genes;

[0044] Using hg19 as a reference genome, the target regions of the FGGY gene were selected from Chr1: 59762781-59762859, the target regions of the LMNA gene were selected from Chr1: 156085007-156085095, the target regions of the REST gene were selected from Chr4: 57773841-57773938, the target regions of the PLOD2 gene were selected from Chr3: 145879404-145879505, and the target regions of the POLR1D gene were selected from... The target region of the NFKBIZ gene is selected from Chr3: 101569020-101569114, the target region of the PRKCB gene is selected from Chr16: 23847529-23847601, the target region of the CCSER1 gene is selected from Chr4: 91048392-91048494, and the target region of the NEFM gene is selected from Chr8: 24772267-24772341.

[0045] Specifically, the classification of adult diffuse gliomas is determined based on the methylation detection results of the target region of the gene.

[0046] Specifically, the analysis unit determines the subtype of adult diffuse glioma based on the ΔCt value of each gene; wherein the ΔCt value is the corresponding gene Ct value minus the internal reference gene Ct value.

[0047] Specifically, if the ΔCt of a certain gene is not greater than a preset value, it is determined to be positive; otherwise, it is determined to be negative. In this embodiment of the invention, the preset value is 7.

[0048] The results were interpreted, and the methylation detection results of each gene target region were represented by a fraction S. ΔCt≤7 was positive, with a value of 1; ΔCt>7 was negative, with a value of 0; no methylation marker was detected, which was also negative, with a value of 0.

[0049] The classification rules for methylation markers are as follows:

[0050] (1) First, the methylation detection results of the target regions of the POLR1D and NFKBIZ genes were analyzed, and MEscore1=S POLR1D +S NFKBIZ When all methylation test results are positive, i.e. MEscore1=2, the sample is identified as IDH mutant; otherwise, the sample is identified as glioblastoma, IDH wild type.

[0051] (2) When the sample is identified as IDH mutant, the methylation detection results of the target regions of the REST, PLOD2, PLOD2, and LMNA genes are analyzed, and the scores are calculated according to the following formula: MEscore2=S REST +S PLOD2 - S FGGY - S LMNA When MEscore2≥1, the sample is identified as oligodendroglioma, with IDH mutation and 1p19q co-deletion; otherwise, the sample is identified as astrocytoma, with IDH mutation and no 1p19q co-deletion.

[0052] (3) When the sample is identified as glioblastoma, IDH wild-type, the methylation detection results of the target regions of PRKCB, CCSER1, and NEFM genes are analyzed, and the score is calculated according to the following formula: MEscore3=S PRKCB + S CCSER1 + S NEFM When MEscore3≥2, the sample is identified as glioblastoma carrying molecular characteristics, IDH wild type; otherwise, the sample is identified as glioblastoma with only pathological characteristics, IDH wild type.

[0053] Example 1

[0054] qPCR detection of biomarker methylation.

[0055] Includes the following steps:

[0056] I. Tissue DNA extraction;

[0057] DNA was extracted from frozen tissue samples using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany, 51304). The extracted DNA was quantified using the Qubit dsDNA HS Assay kit (Life Technologies; Carlsbad, CA, USA) on a Qubit 3.0 fluorometer.

[0058] II. Bismuth transformation of genomic DNA;

[0059] Genomic DNA was sulfite-converted and purified using the EZ DNA Methylation Kit (Zymo Research, D5031).

[0060] III. Methylation qPCR detection;

[0061] 3.1 Instruments and reagents;

[0062] The reagents and instruments used for qPCR detection were: KAPA PROBE FAST qPCR Kit (Roche, KK4702) and qPCR instrument (Tianlong Technology, Gentier 96R).

[0063] 3.2 Primers;

[0064] The primer and probe sequences are shown in Table 1.

[0065] 3.3 qPCR reaction;

[0066] The DNA treated in step two was amplified by fluorescent PCR using the methylation primer and probe combinations described in Table 1 to obtain the Ct values ​​of the target gene and the internal reference gene. The reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0067] Table 2 qPCR reaction system

[0068] Table 3 qPCR reaction procedure

[0069] Note: * indicates fluorescence collection.

[0070] Example 2

[0071] Based on a retrospective cohort, the methylation typing method was determined.

[0072] I. Queue Overview;

[0073] The retrospective cohort included 110 cases of adult diffuse glioma collected between 2010 and 2016. In accordance with WHO CNS 5, all frozen tissue samples underwent guideline-recommended molecular testing: IDH1 / 2, TERT, and H3 mutation detection were performed using the "Five-Item Detection for Gliomas" conducted by Beijing Genecast Technology Co., Ltd.; EGFR amplification, 1p19 co-deletion, and chr7+ / chr10- detection were performed using "Whole Genome Low-Depth Sequencing" conducted by Beijing Genecast Technology Co., Ltd.; and genotyping was performed based on the test results. Specific sample numbers and genotyping details are shown in Table 4.

[0074] Table 4. Overview of the retrospective cohort sample

[0075] II. Determination of methylation qPCR typing method;

[0076] This invention was used to detect methylation biomarkers in 110 samples from a retrospective cohort. qPCR results were analyzed using Tianlong's Gentier 96R software. ΔCt = target gene Ct - internal reference gene Ct. A threshold of ΔCt ≤ 7 was used to interpret the methylation results for each biomarker: ΔCt ≤ 7 was considered positive, otherwise negative (ΔCt > 7 or no methylation biomarker detected). The methylation results for the nine biomarkers are shown below. Figure 2 .

[0077] The methylation detection results for each gene target region are represented by a score S, with a positive value of 1 and a negative value of 0. The classification rules for adult diffuse gliomas based on methylation markers are determined as follows:

[0078] (1) Analyze the methylation detection results of the two markers, POLR1D and NFKBIZ. Calculate the score (0 or 1) of the methylation detection results of the two markers according to the following formula: MEscore1 = S POLR1D +S NFKBIZ When MEscore1=2 (i.e. both markers are positive), the sample is identified as IDH mutant; otherwise, it is glioblastoma, IDH wild-type.

[0079] (2) When the sample is identified as IDH mutant, the methylation detection results of four markers, REST, PLOD2, FGGY, and LMNA, are analyzed. The methylation detection results (0 or 1) of the four markers are calculated according to the following formula: MEscore2=S REST +S PLOD2 - S FGGY - S LMNA When MEscore2≥1, the sample is identified as oligodendroglioma, with IDH mutation and 1p19q co-deletion; otherwise, it is astrocytoma, with IDH mutation and no 1p19q co-deletion.

[0080] (3) When the sample is identified as glioblastoma, IDH wild-type, the methylation detection results of three markers, PRKCB, CCSER1, and NEFM, are analyzed. The methylation detection results of the three markers (0 or 1) are calculated according to the following formula: MEscore3=S PRKCB + S CCSER1 + S NEFM When MEscore 3 ≥ 2 (i.e., two or more markers are positive), the sample is identified as glioblastoma carrying molecular characteristics (having one or more molecular characteristics: TERT promoter mutation, +7 / -10, EGFR amplification), IDH wild type; otherwise, it is glioblastoma with only pathological characteristics, IDH wild type.

[0081] According to existing research, there is a difference in survival between patients with glioblastoma carrying molecular features and those with glioblastoma only having pathological features.

[0082] According to this rule, the 110 samples were classified, as shown in Table 5. The markers showed 100% distinguishing ability between IDH mutant and IDH wild-type tumors; for IDH mutant astrocytomas and oligodendrogliomas, the markers also showed 100% distinguishing ability. For glioblastomas with TERT+ or EGFR amplification or +7 / -10 molecular characteristics, the markers showed 100% sensitivity.

[0083] Table 5. Retrospective cohort methylation typing results

[0084] Where A represents astrocytoma, IDH mutant type without 1p / 19q co-deletion; O represents oligodendroglioma, IDH mutant type with 1p / 19q co-deletion; C represents glioblastoma with molecular characteristics, IDH wild type; D represents glioblastoma with only pathological characteristics, IDH wild type; - indicates that the methylation marker was not detected.

[0085] Example 3

[0086] Based on a prospective cohort, the methylation qPCR typing method was validated.

[0087] I. Queue Overview;

[0088] The prospective cohort included 89 adult patients with diffuse gliomas collected between July and December 2021. Molecular tests (IDH1 / 2, TERT, H3, 1p19q, EGFRamp, +7 / -10) recommended by the WHO CNS 5 were performed on all cases using the same methods as in the retrospective cohort, and the subtyping was confirmed. Specific sample sizes and subtyping details are shown in Table 6.

[0089] Table 6 Queue Samples

[0090] II. Validation results of methylation qPCR typing method;

[0091] The methylation biomarker genotyping rules determined in this invention were used to validate a prospective cohort. The methylation results of the nine biomarkers are shown below. Figure 3The typing results are shown in Table 7. The markers showed 100% distinguishing ability between IDH mutant and IDH wild-type tumors; for astrocytomas and oligodendrogliomas within the IDH mutant group, the markers showed 100% distinguishing ability; and for glioblastomas within the IDH wild-type group with TERT promoter mutations, EGFR amplification, or +7 / -10 molecular characteristics, the markers showed 100% sensitivity.

[0092] Table 7. Prospective cohort methylation typing results

[0093] Where A represents astrocytoma, IDH mutant type without 1p / 19q co-deletion; O represents oligodendroglioma, IDH mutant type with 1p / 19q co-deletion; C represents glioblastoma with molecular characteristics, IDH wild type; D represents glioblastoma with only pathological characteristics, IDH wild type; - indicates that the methylation marker was not detected.

[0094] Example 4

[0095] Comparison of detection results for different biomarkers.

[0096] To verify the impact of different markers and the number of markers selected on the detection results, as typical examples, different numbers of markers were selected for methylation marker detection and genotyping result determination according to the methods in Examples 1 and 2. Specific marker combinations are shown in Table 8, and genotyping results are shown in Tables 10-11. When the number of markers was increased to 10, the genotyping performance remained unchanged; when the number of markers was reduced to 8, the genotyping performance decreased. Specifically, in the retrospective cohort, the genotyping performance remained unchanged, but in the prospective cohort, 7 samples (samples 31, 32, 33, 35, 40, 42, and 43) were incorrectly classified as type A for type O. This indicates that reducing the number of markers resulted in poor and unstable genotyping results. Therefore, a combination of 9 markers is optimal. It should be noted that the combinations and selection numbers of other markers follow the same pattern.

[0097] The rules for determining the combination of the three biomarkers are the same as those for (1) and (3) in the classification rules for adult diffuse glioma in Example 2. Only when the sample is identified as IDH mutant, the combination of the three biomarkers is determined according to the following rules:

[0098] 8 combinations of symbols:

[0099] (2) When the sample is identified as IDH mutant, the methylation detection results of the target regions of the REST, FGGY, and LMNA genes are analyzed, and the score is calculated according to the following formula: MEscore2=S REST- S FGGY -S LMNA When MEscore2≥1, the sample is identified as oligodendroglioma, with IDH mutation and 1p19q co-deletion; otherwise, the sample is identified as astrocytoma, with IDH mutation and no 1p19q co-deletion.

[0100] 9 combinations of symbols:

[0101] (2) When the sample is identified as IDH mutant, the methylation detection results of the target regions of the REST, PLOD2, FGGY, and LMNA genes are analyzed, and the scores are calculated according to the following formula: MEscore2=S REST +S PLOD2 - S FGGY -S LMNA When MEscore2≥1, the sample is identified as oligodendroglioma, with IDH mutation and 1p19q co-deletion; otherwise, the sample is identified as astrocytoma, with IDH mutation and no 1p19q co-deletion.

[0102] 10 combinations of symbols:

[0103] (2) When the sample is identified as IDH mutant, the methylation detection results of the target regions of ATL3, REST, PLOD2, FGGY, and LMNA genes are analyzed. The primer and probe information of the ATL3 gene is shown in Table 9. The score is calculated according to the following formula: MEscore2 = S ATL3 + S REST +S PLOD2 - S FGGY -S LMNA When MEscore2≥1, the sample is identified as oligodendroglioma, with IDH mutation and 1p19q co-deletion; otherwise, the sample is identified as astrocytoma, with IDH mutation and no 1p19q co-deletion.

[0104] Table 8 Different combinations of markers

[0105] Table 9 BT5 marker primer information

[0106] Table 10. Results of different biomarker combinations in the retrospective cohort.

[0107] Table 11 Detection results of different biomarker combinations in the prospective cohort

[0108] Where A represents astrocytoma, IDH mutant type without 1p / 19q co-deletion; O represents oligodendroglioma, IDH mutant type with 1p / 19q co-deletion; C represents glioblastoma with molecular characteristics, IDH wild type; D represents glioblastoma with only pathological characteristics, IDH wild type; - indicates that the methylation marker was not detected.

[0109] In summary, this invention, which uses methylation markers for the classification of adult diffuse gliomas, has the following significant advantages:

[0110] 1) Simple and efficient detection: Using a single DNA methylomics detection method, only 9 specific methylation markers need to be analyzed to complete the typing. Compared with the multi-platform combined detection recommended by the guidelines (such as IDH mutation, 1p19q co-deletion, TERT promoter mutation, EGFR amplification, etc.), the operation process is greatly simplified, which is especially suitable for clinical scenarios with limited tissue sample volume.

[0111] 2) Low cost and low technical threshold: The detection method based on qPCR technology is significantly cheaper than Illumina methylation chips, and does not rely on complex instruments or professional analysis platforms, making it easier to carry out routinely in hospitals and reducing the technical and hardware requirements of medical institutions.

[0112] 3) Fast and easy to localize: The detection cycle is short, overcoming the limitation of methylation chips requiring a two-week cycle. At the same time, it eliminates the dependence on overseas reference datasets and pre-built models, avoids the compliance risks of cross-border data transmission, and achieves fully autonomous and localized deployment.

[0113] 4) Clinical Applicability and Stability: By using a precisely selected combination of methylation markers, the inconsistencies in results across multiple platforms (such as differences in FISH and NGS interpretation of 1p19q) are avoided, improving the reliability of the subtyping results and providing efficient and stable molecular diagnostic support for clinical decision-making. This invention, with its single-technology platform, low cost, and rapid and convenient solution, solves the current technical bottlenecks in glioma molecular subtyping. It can assist in the molecular subtyping of adult diffuse gliomas, demonstrating significant clinical application value and potential for widespread adoption.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A combination of DNA methylation markers for the subtyping of adult diffuse gliomas for non-disease diagnostic and therapeutic purposes, characterized in that, include: Methylation sites in the target regions of the FGGY, LMNA, REST, PLOD2, POLR1D, NFKBIZ, PRKCB, CCSER1, and NEFM genes; Using hg19 as a reference genome, the target regions of the FGGY gene were selected from Chr1: 59762781-59762859, the target regions of the LMNA gene were selected from Chr1: 156085007-156085095, the target regions of the REST gene were selected from Chr4: 57773841-57773938, the target regions of the PLOD2 gene were selected from Chr3: 145879404-145879505, and the target regions of the POLR1D gene were selected from... The target region of the NFKBIZ gene is selected from Chr3: 101569020-101569114, the target region of the PRKCB gene is selected from Chr16: 23847529-23847601, the target region of the CCSER1 gene is selected from Chr4: 91048392-91048494, and the target region of the NEFM gene is selected from Chr8: 24772267-24772341.

2. A reagent kit, characterized in that, include: An upstream primer and a downstream primer, wherein the upstream primer and the downstream primer are used to detect the methylation marker as described in claim 1; The upstream primers are shown in SEQ ID No: 1-9; The downstream primers are shown in SEQ ID No: 10-18.

3. The reagent kit according to claim 2, characterized in that, It also includes probes corresponding to the upstream and downstream primers, as shown in SEQ ID No: 19-27.

4. The reagent kit according to claim 2, characterized in that, It also includes an internal reference gene, which is the GAPDH gene. The primers for the GAPDH gene are shown in SEQ ID No: 28-29, and the probe for the GAPDH gene is shown in SEQ ID No:

30.

5. A device for typing diffuse gliomas in adults, characterized in that, include: The detection unit uses the kit described in any one of claims 2-4 to detect the methylation level of the target region of the gene and obtain the detection result. An analysis unit that determines the subtype of adult diffuse glioma based on the detection results.

6. The apparatus according to claim 5, characterized in that, The detection unit includes a real-time PCR instrument.

7. The apparatus according to claim 5, characterized in that, The analysis unit determines the adult diffuse glioma subtype based on the ΔCt value of each gene; The ΔCt value is the Ct value of the corresponding gene minus the Ct value of the internal reference gene.

8. The apparatus according to claim 7, characterized in that, If the ΔCt of the gene is not greater than a preset value, it is considered positive; otherwise, it is considered negative.

9. The apparatus according to claim 8, characterized in that, The methylation detection results of the target region of the gene are represented by a fraction S, with a positive value of 1 and a negative value of 0. MEscore1=S POLR1D +S NFKBIZ When MEscore1=2, the sample is identified as IDH mutant; otherwise, the sample is identified as glioblastoma, IDH wild type. When a sample is identified as an IDH mutant, methylation detection results of the target regions of the REST, PLOD2, FGGY, and LMNA genes are analyzed, with MEscore2=S. REST +S PLOD2 - S FGGY -S LMNA When MEscore2≥1, the sample is identified as oligodendroglioma, with IDH mutation and 1p19q co-deletion; otherwise, the sample is identified as astrocytoma, with IDH mutation and no 1p19q co-deletion. When the sample was identified as glioblastoma, IDH wild-type, methylation detection results of the target regions of PRKCB, CCSER1, and NEFM genes were analyzed, with MEscore3=S. PRKCB + S CCSER1 + S NEFM When MEscore3≥2, the sample is identified as glioblastoma carrying molecular characteristics, IDH wild type; otherwise, the sample is identified as glioblastoma with only pathological characteristics, IDH wild type.

10. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores one or more programs, which can be executed by one or more processors to perform the following method: Analyze the results detected by the kit described in any one of claims 2-4; Calculate the ΔCt of the corresponding gene. If the ΔCt of the gene is not greater than the preset value, it is judged as positive; otherwise, it is judged as negative. The methylation detection results of the target region of the gene are represented by a fraction S, with a positive value of 1 and a negative value of 0. MEscore1=S POLR1D +S NFKBIZ When MEscore1=2, the sample is identified as IDH mutant; otherwise, the sample is identified as glioblastoma, IDH wild type. When a sample is identified as an IDH mutant, methylation detection results of the target regions of the REST, PLOD2, FGGY, and LMNA genes are analyzed, with MEscore2=S. REST +S PLOD2 - S FGGY -S LMNA When MEscore2≥1, the sample is identified as oligodendroglioma, with IDH mutation and 1p19q co-deletion; otherwise, the sample is identified as astrocytoma, with IDH mutation and no 1p19q co-deletion. When the sample was identified as glioblastoma, IDH wild-type, methylation detection results of the target regions of PRKCB, CCSER1, and NEFM genes were analyzed, with MEscore3=S. PRKCB + S CCSER1 + S NEFM When MEscore3≥2, the sample is identified as glioblastoma carrying molecular characteristics, IDH wild type; otherwise, the sample is identified as glioblastoma with only pathological characteristics, IDH wild type.

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