[go: up one dir, main page]

WO2024027796A1 - Utilisation d'un marqueur dans le diagnostic du cancer du sein ou la prédiction de risques de cancer du sein - Google Patents

Utilisation d'un marqueur dans le diagnostic du cancer du sein ou la prédiction de risques de cancer du sein Download PDF

Info

Publication number
WO2024027796A1
WO2024027796A1 PCT/CN2023/111009 CN2023111009W WO2024027796A1 WO 2024027796 A1 WO2024027796 A1 WO 2024027796A1 CN 2023111009 W CN2023111009 W CN 2023111009W WO 2024027796 A1 WO2024027796 A1 WO 2024027796A1
Authority
WO
WIPO (PCT)
Prior art keywords
marker
target
methylation
breast cancer
combination
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2023/111009
Other languages
English (en)
Chinese (zh)
Inventor
孙津
马成城
徐敏杰
刘轶颖
苏志熙
刘蕊
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Singlera Genomics China Ltd
Original Assignee
Singlera Genomics China Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN202210931191.8A external-priority patent/CN117587121A/zh
Priority claimed from CN202210931401.3A external-priority patent/CN117660634A/zh
Priority claimed from CN202210931193.7A external-priority patent/CN117660633A/zh
Application filed by Singlera Genomics China Ltd filed Critical Singlera Genomics China Ltd
Publication of WO2024027796A1 publication Critical patent/WO2024027796A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • This application relates to the field of molecular biomedicine technology. Specifically, this application relates to the use of markers in diagnosing breast cancer or predicting breast cancer risk.
  • Breast cancer is a malignant cancer with the highest morbidity and mortality among women. 5%-10% of breast cancer patients are caused by inherited gene mutations, and other cancers are mainly caused by factors such as the environment (Feng Y, Spezia M, Huang S, et al. Breast cancer development and progression: Risk factors, cancer stem cells, signaling pathways, genomics, and molecular pathogenesis. Genes Dis. 2018; 5: 77-106.).
  • the current clinical breast cancer diagnosis strategies are: 1. Breast digital examination and physical examination by a doctor; 2. Breast ultrasound and mammography; 3. Fine-needle aspiration tissue biopsy, etc.
  • mammography can detect breast tumors and find signs of malignant tumors such as breast microcalcifications, and is often used for early screening of high-risk women (Oeffinger KC, Fontham ET, Etzioni R, et al. Breast Cancer Screening for Women at Average Risk: 2015 Guideline Update From the American Cancer Society. JAMA. 2015;314:1599-614.).
  • the accuracy of mammography detection is closely related to breast structure.
  • Liquid biopsy is a non-invasive detection technology based on blood or other body fluids. It has the advantages of safe, non-invasive sampling, high efficiency and convenience, and has been gradually used in the detection of various diseases.
  • Research has found that body fluids contain a large amount of free DNA (cfDNA) released by tissues or cells.
  • the body fluids of cancer patients also contain free tumor DNA (ctDNA) released by cancer tissues into body fluids.
  • cfDNA free DNA
  • ctDNA free tumor DNA
  • Its genomic characteristics include mutations, fragmentation length distribution, terminal Motifs, DNA methylation, etc. can be used as characteristic indicators for early cancer diagnosis (Lo YMD, Han DSC, Jiang P, et al. Epigenetics, fragmentomics, and topology of cell-free DNA in liquid biopsies. Science. 2021; 372.).
  • DNA methylation has the advantages of low detection limit, high sensitivity, and high specificity, and has been successfully used in the diagnosis of various cancers.
  • methylation levels of 6 methylation markers were used to construct colorectal cancer plasma.
  • the machine learning diagnostic model can achieve 92% specificity and 86% sensitivity (Cai G, Cai M, Feng Z, et al. A Multilocus Blood-Based Assay Targeting Circulating Tumor DNA Methylation Enables Early Detection and Early Relapse Prediction of Colorectal Cancer.
  • the inventors screened a large number of markers and found that the markers of the present invention can diagnose breast cancer or predict the risk of breast cancer with high sensitivity, specificity and low cost. Based on the markers of the present invention, breast cancer patients and healthy people can be effectively distinguished.
  • the present invention provides 79 cfDNA methylation markers and establishes a diagnostic model of the relationship between methylation marker methylation levels and breast cancer.
  • This model has non-invasive detection, safe and convenient detection, and high throughput. , the advantage of high detection accuracy.
  • the invention relates to the use of reagents in the preparation of kits or microarrays for diagnosing breast cancer or predicting the risk of breast cancer in an individual, characterized in that said reagents are used for detection in a sample isolated from said individual Methylation level of at least one target region of at least one marker selected from any of the following groups:
  • a methylation level of at least one target region of one or more markers equal to or above the threshold value, compared to a corresponding threshold value, indicates that the individual has breast cancer or is at risk for breast cancer, and wherein the target region Contains at least one CpG dinucleotide sequence.
  • the methylation is CpG methylation.
  • the reagent is a reagent selected from:
  • a substance that hybridizes to or amplifies at least one target region of said marker such as an oligonucleotide primer or probe
  • a bisulfite reagent or a methylation-sensitive restriction enzyme reagent that distinguishes between methylated and unmethylated within at least one target region of the marker dinucleotides, such as methylated and unmethylated CpG dinucleotides.
  • the oligonucleotide primer or probe is complementary or identical to an at least 9 base long fragment of at least one target region of the marker.
  • the marker is TTLL10. In some embodiments, the marker is EPS8L3. In some embodiments, the marker is IRF2BP2. In some embodiments, the marker is FAM150B. In some embodiments, the marker is ID2. In some embodiments, the marker is TERT. In some embodiments, the marker is PITX1. In some embodiments, the marker is KCNMB1. In some embodiments, the marker is BEND6. In a In some embodiments, the marker is ELN. In some embodiments, the marker is CPXM2. In some embodiments, the marker is TH. In some embodiments, the marker is C1QTNF9. In some embodiments, the marker is CARKD. In some embodiments, the marker is TMEM179.
  • the marker is SPNS1. In some embodiments, the marker is MYO15B. In some embodiments, the marker is DNM2. In some embodiments, the marker is EPHX3. In some embodiments, the marker is PSG8. In some embodiments, the marker is SLCO4A1. In some embodiments, the marker is TNFRSF6B.
  • the marker is SKI. In some embodiments, the marker is PRDM16. In some embodiments, the marker is PIAS3. In some embodiments, the marker is SLC10A4. In some embodiments, the marker is CXXC5. In some embodiments, the marker is NR2E1. In some embodiments, the marker is MPC1. In some embodiments, the marker is HOXA13. In some embodiments, the marker is LZTS 1. In some embodiments, the marker is CHD7. In some embodiments, the marker is ANKRD20A1. In some embodiments, the marker is CACNA1B. In some embodiments, the marker is ACVRL1. In some embodiments, the marker is CCNA1. In some embodiments, the marker is RNASEH2B.
  • the marker is SNX20. In some embodiments, the marker is TBCD. In some embodiments, the marker is PIP5K1C. In some embodiments, the marker is ZBTB7A. In some embodiments, the marker is DNASE2. In some embodiments, the marker is TSHZ3. In some embodiments, the marker is WISP2.
  • the marker is WRAP73. In some embodiments, the marker is C2CD4D. In some embodiments, the marker is CCDC181. In some embodiments, the marker is RNF144A. In some embodiments, the marker is SIX2. In some embodiments, the marker is NRXN1. In some embodiments, the marker is MEIS1. In some embodiments, the marker is LBX2. In some embodiments, the marker is AMT. In some embodiments, the marker is ITIH4. In some embodiments, the marker is TRH. In some embodiments, the marker is SHOX2. In some embodiments, the marker is DGKG. In some embodiments, the marker is RPL9. In some embodiments, the marker is PFN3. In some embodiments, the marker is FOXC1.
  • the marker is LY86. In some embodiments, the marker is SLC35F1. In some embodiments, the marker is LRRC4. In some embodiments, the marker is PDLIM2. In some embodiments, the marker is PAX2. In some embodiments, the marker is MVK. In some embodiments, the marker is DTX1. In some embodiments, the marker is RBM19. In some embodiments, the marker is GCH1. In some embodiments, the marker is OTX2. In some embodiments, the marker is ZSCAN10. In some embodiments, the marker is AHSP. In some embodiments, the marker is NLRC5. In some embodiments, the marker is ASXL3. In some embodiments, the marker is TCF4. In some embodiments, the marker is PLIN3. In some embodiments, the marker is RASAL3. In some embodiments, the marker is CHRNA4.
  • the marker is a combination of markers selected from: i) TTLL10, FAM150B, BEND6, ELN, TMEM179, and MYO15B; or ii) EPS8L3, IRF2BP2, TERT, TH, CARKD, SPNS1, and PSG8.
  • the marker is a combination of markers selected from: i) SKI, PRDM16, LZTS1, CCNA1, PIP5K1C, and WISP2; or ii) PIAS3, CHD7, CACNA1B, ACVRL1, SNX20, TBCD, and ZBTB7A.
  • the marker is a combination of markers selected from: i) ITIH4, FOXC1, PDLIM2, MVK, NLRC5, TCF4, and PLIN3; or ii) RNF144A, SIX2, DGKG, RPL9, LRRC4, and ZSCAN10.
  • the sample is selected from the group consisting of cell lines, histological sections, tissue biopsies, paraffin-embedded tissues, body fluids, and combinations thereof; preferably, the sample is selected from the group consisting of plasma, serum, whole blood, isolated blood cells and combinations thereof; more preferably, the sample is plasma cfDNA or ctDNA.
  • the target region is selected from: region chr1:1095763-1095986, chr1:110334699-110334899, chr1:234845168-234845486, chr2:469568-469933, chr2:8314701-8314901, chr5:1 291139-1291339 ⁇ chr5: 134374689-134374889, chr5: 169805839-169806039, chr6: 56716287-56716518, chr7: 73407894-73408161, chr10: 125650986-125651186, chr11: 22 26052-2226252, chr13: 111277395-111277690, chr13: 24844736-24844936, chr14: 105102434-105102644, chr16: 28984534-28984734, chr17: 73607909-73608115,
  • the target area is selected from: area chr1: 2166118-2166318, chr1: 2978722-2978922, chr1: 145562922-145563122, chr4: 48485417-48485821, chr5: 139076623-139076941, chr6: 10 8488634-108488917, chr6: 166970625-166970825, chr7: 27260117-27260462, chr8: 20375580-20375780, chr8: 61788861-61789200, chr9: 68413067-68413267, chr9: 140683687-140683969, chr12: 5231164 7-52311991, chr13: 37005935-37006328, chr13: 51417486-51417774, chr16: 50715367-50715567, chr17: 80745056-80745446, chr
  • the target area is selected from: area chr1: 3567381-3567648, chr1: 151811354-151811554, chr1: 169396540-169396740, chr2: 7148520-7148720, chr2: 45232498-45232698, chr2: 50 574443-50574739, chr2: 66666356-66666556, chr2: 74731340-74731602, chr3: 49459532-49459732, chr3: 52864771-52864971, chr3: 52865018-52865236, chr3: 129693578-129693796, chr3: 15782502 5-157825225, chr3: 185973717-185973917, chr4: 39448374-39448574, chr5: 176829529-176829796, chr6: 1614911-1615
  • chr16 57025884-57026193, chr18: 31159160-31159360, chr18: 53447617-53447817, chr19: 4912069-4912269, chr19: 15580341-15580719, chr20: 62046355-62046589 or their complementary sequences or processed sequences; or processed sequences of the complementary sequences; or any combination of the aforementioned sequences and/or regions.
  • the invention relates to a kit or a microarray for diagnosing breast cancer or predicting the risk of breast cancer in an individual, characterized in that the kit or microarray contains a method for detecting a gene isolated from said individual.
  • a reagent for the methylation level of at least one target region of at least one marker selected from any of the following groups in the sample:
  • a methylation level of the target region of one or more markers equal to or above the threshold value compared to a corresponding threshold value indicates that the individual has breast cancer or is at risk for breast cancer, and wherein the target region contains at least A CpG dinucleotide sequence.
  • the methylation is CpG methylation.
  • the sample is selected from the group consisting of cell lines, histological sections, tissue biopsies, paraffin-embedded tissues, body fluids, and combinations thereof; preferably, the sample is selected from the group consisting of plasma, serum, whole blood, isolated blood cells and combinations thereof; more preferably, the sample is plasma cfDNA or ctDNA.
  • the reagent is a reagent selected from:
  • a substance that hybridizes to or amplifies at least one target region of the marker such as an oligonucleotide primer or probe, preferably the oligonucleotide primer or probe
  • the needle is complementary or identical to a fragment of at least 9 bases in length of at least one target region of the marker;
  • a bisulfite reagent or a methylation-sensitive restriction enzyme reagent that distinguishes between methylated and unmethylated within at least one target region of the marker dinucleotides, such as methylated and unmethylated CpG dinucleotides.
  • the marker is TTLL10. In some embodiments, the marker is EPS8L3. In some embodiments, the marker is IRF2BP2. In some embodiments, the marker is FAM150B. In some embodiments, the marker is ID2. In some embodiments, the marker is TERT. In some embodiments, the marker is PITX1. In some embodiments, the marker is KCNMB1. In some embodiments, the marker is BEND6. In some embodiments, the marker is ELN. In some embodiments, the marker is CPXM2. In some embodiments, the marker is TH. In some embodiments, the marker is C1QTNF9. In some embodiments, the marker is CARKD. In some embodiments, the marker is TMEM179.
  • the marker is SPNS1. In some embodiments, the marker is MYO15B. In some embodiments, the marker is DNM2. In some embodiments, the marker is EPHX3. In some embodiments, the marker is PSG8. In some embodiments, the marker is SLCO4A1. In some embodiments, the marker is TNFRSF6B.
  • the marker is SKI. In some embodiments, the marker is PRDM16. In some embodiments, the marker is PIAS3. In some embodiments, the marker is SLC10A4. In some embodiments, the marker is CXXC5. In some embodiments, the marker is NR2E1. In some embodiments, the marker is MPC1. In some embodiments, the marker is HOXA13. In some embodiments, the marker is LZTS 1. In some embodiments, the marker is CHD7. In some embodiments, the marker is ANKRD20A1. In some embodiments, the marker is CACNA1B. In some embodiments, the marker is ACVRL1. In some embodiments, the marker is CCNA1. In some embodiments, the marker is RNASEH2B.
  • the marker is SNX20. In some embodiments, the marker is TBCD. In some embodiments, the marker is PIP5K1C. In some embodiments, the marker is ZBTB7A. In some implementations In the scheme, the marker is DNASE2. In some embodiments, the marker is TSHZ3. In some embodiments, the marker is WISP2.
  • the marker is WRAP73. In some embodiments, the marker is C2CD4D. In some embodiments, the marker is CCDC181. In some embodiments, the marker is RNF144A. In some embodiments, the marker is SIX2. In some embodiments, the marker is NRXN1. In some embodiments, the marker is MEIS1. In some embodiments, the marker is LBX2. In some embodiments, the marker is AMT. In some embodiments, the marker is ITIH4. In some embodiments, the marker is TRH. In some embodiments, the marker is SHOX2. In some embodiments, the marker is DGKG. In some embodiments, the marker is RPL9. In some embodiments, the marker is PFN3. In some embodiments, the marker is FOXC1.
  • the marker is LY86. In some embodiments, the marker is SLC35F1. In some embodiments, the marker is LRRC4. In some embodiments, the marker is PDLIM2. In some embodiments, the marker is PAX2. In some embodiments, the marker is MVK. In some embodiments, the marker is DTX1. In some embodiments, the marker is RBM19. In some embodiments, the marker is GCH1. In some embodiments, the marker is OTX2. In some embodiments, the marker is ZSCAN10. In some embodiments, the marker is AHSP. In some embodiments, the marker is NLRC5. In some embodiments, the marker is ASXL3. In some embodiments, the marker is TCF4. In some embodiments, the marker is PLIN3. In some embodiments, the marker is RASAL3. In some embodiments, the marker is CHRNA4.
  • the marker is a combination of markers selected from: i) TTLL10, FAM150B, BEND6, ELN, TMEM179, and MYO15B; or ii) EPS8L3, IRF2BP2, TERT, TH, CARKD, SPNS 1, and PSG8 .
  • the marker is a combination of markers selected from: i) SKI, PRDM16, LZTS1, CCNA1, PIP5K1C, and WISP2; or ii) PIAS3, CHD7, CACNA1B, ACVRL1, SNX20, TBCD, and ZBTB7A.
  • the marker is a combination of markers selected from: i) ITIH4, FOXC1, PDLIM2, MVK, NLRC5, TCF4, and PLIN3; or ii) RNF144A, SIX2, DGKG, RPL9, LRRC4, and ZSCAN10.
  • the target region is selected from: region chr1:1095763-1095986, chr1:110334699-110334899, chr1:234845168-234845486, chr2:469568-469933, chr2:8314701-8314901, chr5:1 291139-1291339 ⁇ chr5: 134374689-134374889, chr5: 169805839-169806039, chr6: 56716287-56716518, chr7: 73407894-73408161, chr10: 125650986-125651186, chr11: 22 26052-2226252, chr13: 111277395-111277690, chr13: 24844736-24844936, chr14: 105102434-105102644, chr16: 28984534-28984734, chr17: 73607909-73608115,
  • the target area is selected from: area chr1: 2166118-2166318, chr1: 2978722-2978922, chr1: 145562922-145563122, chr4: 48485417-48485821, chr5: 139076623-139076941, chr6: 10 8488634-108488917, chr6: 166970625-166970825, chr7: 27260117-27260462, chr8: 20375580-20375780, chr8: 61788861-61789200, chr9: 68413067-68413267, chr9: 140683687-140683969, chr12: 5231164 7-52311991, chr13: 37005935-37006328, chr13: 51417486-51417774, chr16: 50715367-50715567, chr17: 80745056-80745446, chr
  • the target area is selected from: area chr1: 3567381-3567648, chr1: 151811354-151811554, chr1: 169396540-169396740, chr2: 7148520-7148720, chr2: 45232498-45232698, chr2: 50 574443-50574739, chr2: 66666356-66666556, chr2: 74731340-74731602, chr3: 49459532-49459732, chr3: 52864771-52864971, chr3: 52865018-52865236, chr3: 129693578-129693796, chr3: 15782502 5-157825225, chr3: 185973717-185973917, chr4: 39448374-39448574, chr5: 176829529-176829796, chr6: 1614911-1615
  • chr16 57025884-57026193, chr18: 31159160-31159360, chr18: 53447617-53447817, chr19: 4912069-4912269, chr19: 15580341-15580719, chr20: 62046355-62046589 or their complementary sequences or processed sequences; or processed sequences of the complementary sequences; or any combination of the aforementioned sequences and/or regions.
  • the invention relates to a method for diagnosing breast cancer or predicting breast cancer risk in an individual, said method comprising the steps of:
  • step (b) treating the DNA in the biological sample obtained in step (a) with a reagent capable of distinguishing between methylated and unmethylated sites, such as CpG sites, in the DNA, Thus processed DNA is obtained;
  • step (c) pre-amplify at least one target region of at least one target marker in the processed DNA obtained from step (b) using a pool of pre-amplification primers, wherein at least one target region of each target marker A target region is preamplified to obtain at least one preamplification product, and the at least one target marker includes one or more markers selected from any of the following groups:
  • said target region comprises at least one CpG dinucleotide sequence
  • step (d) Detecting a methylation template of at least one target region of at least one target marker in step (b) or step (c), the at least one target marker comprising one selected from any of the following groups or multiple markers:
  • step (e) Compare the methylation level of at least one target region of each target marker in step (d) with the corresponding threshold value, respectively, wherein at least one target region of one or more target markers is relative to its corresponding threshold value Having a methylation level equal to or above a threshold indicates that the individual has breast cancer or is at risk for breast cancer.
  • the reagent is a bisulfite reagent or a methylation-sensitive restriction enzyme reagent that distinguishes within at least one target region of the marker Methylated and unmethylated dinucleotides, such as methylated and unmethylated CpG dinucleotides.
  • step (c) amplification is performed using substances, such as oligonucleotide primers, that amplify at least one target region of the marker.
  • the oligonucleotide primer is complementary or identical to an at least 9 base long fragment of at least one target region of the marker.
  • the oligonucleotide primer is selected from SEQ ID NO: 23-66.
  • detection is performed using a substance, such as a probe, that hybridizes to at least one target region of the marker.
  • the probe is complementary or identical to an at least 9 base long fragment of at least one target region of the marker.
  • the marker is TTLL10. In some embodiments, the marker is EPS8L3. In some embodiments, the marker is IRF2BP2. In some embodiments, the marker is FAM150B. In some embodiments, The marker is ID2. In some embodiments, the marker is TERT. In some embodiments, the marker is PITX1. In some embodiments, the marker is KCNMB1. In some embodiments, the marker is BEND6. In some embodiments, the marker is ELN. In some embodiments, the marker is CPXM2. In some embodiments, the marker is TH. In some embodiments, the marker is C1QTNF9. In some embodiments, the marker is CARKD. In some embodiments, the marker is TMEM179.
  • the marker is SPNS1. In some embodiments, the marker is MYO15B. In some embodiments, the marker is DNM2. In some embodiments, the marker is EPHX3. In some embodiments, the marker is PSG8. In some embodiments, the marker is SLCO4A1. In some embodiments, the marker is TNFRSF6B.
  • the marker is SKI. In some embodiments, the marker is PRDM16. In some embodiments, the marker is PIAS3. In some embodiments, the marker is SLC10A4. In some embodiments, the marker is CXXC5. In some embodiments, the marker is NR2E1. In some embodiments, the marker is MPC1. In some embodiments, the marker is HOXA13. In some embodiments, the marker is LZTS 1. In some embodiments, the marker is CHD7. In some embodiments, the marker is ANKRD20A1. In some embodiments, the marker is CACNA1B. In some embodiments, the marker is ACVRL1. In some embodiments, the marker is CCNA1. In some embodiments, the marker is RNASEH2B.
  • the marker is SNX20. In some embodiments, the marker is TBCD. In some embodiments, the marker is PIP5K1C. In some embodiments, the marker is ZBTB7A. In some embodiments, the marker is DNASE2. In some embodiments, the marker is TSHZ3. In some embodiments, the marker is WISP2.
  • the marker is WRAP73. In some embodiments , the marker is C2CD4D. In some embodiments, the marker is CCDC181. In some embodiments, the marker is RNF144A. In some embodiments, the marker is SIX2. In some embodiments, the marker is NRXN1. In some embodiments, the marker is MEIS1. In some embodiments, the marker is LBX2. In some embodiments, the marker is AMT. In some embodiments, the marker is ITIH4. In some embodiments, the marker is TRH. In some embodiments, the marker is SHOX2. In some embodiments, the marker is DGKG. In some embodiments, the marker is RPL9. In some embodiments, the marker is PFN3. In some embodiments, the marker is FOXC1.
  • the marker is LY86. In some embodiments, the marker is SLC35F1. In some embodiments, the marker is LRRC4. In some embodiments, the marker is PDLIM2. In some embodiments, the marker is PAX2. In some embodiments, the marker is MVK. In some embodiments, the marker is DTX1. In some embodiments, the marker is RBM19. In some embodiments, the marker is GCH1. In some embodiments, the marker is OTX2. In some embodiments, the marker is ZSCAN10. In some embodiments, the marker is AHSP. In some embodiments, the marker is NLRC5. In some embodiments, the marker is ASXL3. In some embodiments, the marker is TCF4. In some embodiments, the marker is PLIN3. In some embodiments, the marker is RASAL3. In some embodiments, the marker is CHRNA4.
  • the marker is a combination of markers selected from: i) TTLL10, FAM150B, BEND6, ELN, TMEM179, and MYO15B; or ii) EPS8L3, IRF2BP2, TERT, TH, CARKD, SPNS 1, and PSG8 .
  • the marker is a combination of markers selected from: i) SKI, PRDM16, LZTS1, CCNA1, PIP5K1C, and WISP2; or ii) PIAS3, CHD7, CACNA1B, ACVRL1, SNX20, TBCD, and ZBTB7A.
  • the marker is a combination of markers selected from: i) ITIH4, FOXC1, PDLIM2, MVK, NLRC5, TCF4, and PLIN3; or ii) RNF144A, SIX2, DGKG, RPL9, LRRC4, and ZSCAN10.
  • the sample is selected from the group consisting of cell lines, histological sections, tissue biopsies, paraffin-embedded tissues, body fluids, and combinations thereof; preferably, the sample is selected from the group consisting of plasma, serum, whole blood, isolated blood cells and combinations thereof; more preferably, the sample is plasma cfDNA or ctDNA.
  • the detection uses gene sequencing, PCR (eg, fluorescent PCR), FISH, immunohistochemistry, ELISA, Western or flow cytometry as the detection method.
  • PCR eg, fluorescent PCR
  • FISH fluorescent PCR
  • immunohistochemistry e.g., immunohistochemistry
  • ELISA Western or flow cytometry
  • the target region is selected from: region chr1:1095763-1095986, chr1:110334699-110334899, chr1:234845168-234845486, chr2:469568-469933, chr2:8314701-8314901, chr5:1 291139-1291339 ⁇ chr5: 134374689-134374889, chr5: 169805839-169806039, chr6: 56716287-56716518, chr7: 73407894-73408161, chr10: 125650986-125651186, chr11: 22 26052-2226252, chr13: 111277395-111277690, chr13: 24844736-24844936, chr14: 105102434-105102644, chr16: 28984534-28984734, chr17: 73607909-73608115,
  • the target area is selected from: area chr1: 2166118-2166318, chr1: 2978722-2978922, chr1: 145562922-145563122, chr4: 48485417-48485821, chr5: 139076623-139076941, chr6: 10 8488634-108488917, chr6: 166970625-166970825, chr7: 27260117-27260462, chr8: 20375580-20375780, chr8: 61788861-61789200, chr9: 68413067-68413267, chr9: 14068368 7-140683969, chr12: 52311647-52311991, chr13: 37005935-37006328, chr13: 51417486-51417774, chr16: 50715367-50715567, chr17: 80745056-80745446, ch
  • the target area is selected from: area chr1: 3567381-3567648, chr1: 151811354-151811554, chr1: 169396540-169396740, chr2: 7148520-7148720, chr2: 45232498-45232698, chr2: 50 574443-50574739, chr2: 66666356-66666556, chr2: 74731340-74731602, chr3: 49459532-49459732, chr3: 52864771-52864971, chr3: 52865018-52865236, chr3: 129693578-129693796, chr3: 15782502 5-157825225, chr3: 185973717-185973917, chr4: 39448374-39448574, chr5: 176829529-176829796, chr6: 1614911-1615
  • chr16 57025884-57026193, chr18: 31159160-31159360, chr18: 53447617-53447817, chr19: 4912069-4912269, chr19: 15580341-15580719, chr20: 62046355-62046589 or their complementary sequences or processed sequences; or processed sequences of the complementary sequences; or any combination of the aforementioned sequences and/or regions.
  • Figure 1 shows the breast cancer marker screening process.
  • Figure 2A shows the methylation levels of the selected 22 markers in the training set and the test set
  • Figure 2B shows the methylation levels of the selected 22 markers in the training set and the test set
  • Figure 2C shows the selected Methylation levels of 35 markers in the training set and test set.
  • Figure 3A shows the methylation level of Seq ID NO: 14 in the training set and the test set
  • Figure 3B shows the methylation level of Seq ID NO: 75 in the training set and the test set
  • Figure 3C shows the methylation level of Seq ID NO: Methylation levels of 152 in the training set and test set
  • Figures 4A, 4B and 4C show AllModel model prediction score distributions.
  • Figure 5A, Figure 5B and Figure 5C show the ROC curves of the AllModel model in the training set and test set.
  • Figures 6A, 6B and 6C show Sub1 model prediction score distributions.
  • Figure 7A, Figure 7B and Figure 7C show the ROC curves of the Sub1 model in the training set and test set.
  • Figures 8A, 8B and 8C show Sub2 model prediction score distributions.
  • Figure 9A, Figure 9B and Figure 9C show the ROC curves of the Sub2 model in the training set and test set.
  • the present invention relates to the relationship between the methylation levels of newly discovered markers and breast cancer.
  • the markers described herein provide methods for diagnosing breast cancer or assessing breast cancer risk in an individual. Therefore, one embodiment of the invention represents an improvement in markers suitable for diagnosing breast cancer or assessing breast cancer risk.
  • the newly discovered markers of the present invention may be combined with one or more other breast cancer markers known in the art (e.g., CEA, CA 15-3, CA 125, Ki-67, HER-2 , ER, PR, etc.) and/or Routine examination methods such as digital breast examination in conjunction with a physician's physical examination, breast ultrasound and mammography, fine needle aspiration tissue biopsy, etc., for example to diagnose breast cancer or assess breast cancer risk in an individual or to prepare preparations for this purpose kits and/or microarrays.
  • other breast cancer markers known in the art e.g., CEA, CA 15-3, CA 125, Ki-67, HER-2 , ER, PR, etc.
  • Routine examination methods such as digital breast examination in conjunction with a physician's physical examination, breast ultrasound and mammography, fine needle aspiration tissue biopsy, etc., for example to diagnose breast cancer or assess breast cancer risk in an individual or to prepare preparations for this purpose kits and/or microarrays.
  • sample means material known or suspected to express or contain a marker as described herein.
  • Samples may be derived from biological sources ("biological samples") such as tissues (e.g. biopsy samples), extracts or cell cultures including cells (e.g. tumor cells), cell lysates and biological or physiological fluids, e.g. Whole blood, plasma, serum, saliva, cerebral spinal fluid, sweat, urine, breast milk, peritoneal fluid, etc. Samples obtained from the source or after pretreatment to improve sample characteristics (eg preparation of plasma from blood, etc.) can be used directly.
  • the sample is a human physiological fluid, such as human plasma.
  • the sample is a biopsy sample such as tumor tissue or cells obtained by tissue examination.
  • Samples that may be analyzed in accordance with the present invention include polynucleotides of clinical origin.
  • the target polynucleotide may comprise DNA or RNA, especially DNA, especially cell-free DNA such as extracellular cell-free DNA.
  • the sample is plasma cfDNA or ctDNA.
  • the target polynucleotide or a substance that hybridizes or amplifies the target polynucleotide can be detectably labeled on one or more nucleotides using methods known in the art.
  • Detectable labels may be, but are not limited to, luminescent labels, fluorescent labels, bioluminescent labels, chemiluminescent labels, radioactive labels, and colorimetric labels.
  • the term "marker” refers to a nucleic acid, gene region or methylation site of interest: its methylation level or the score of a computational model based on the methylation level (e.g., using a machine learning model such as a logistic In the case of regression models, the AUC of the ROC curve indicates a breast cancer diagnosis or a high risk of breast cancer.
  • a gene should be considered to include all its transcript variants and all of its promoters and regulatory elements. As understood by those skilled in the art, certain genes are known to exhibit allelic variation or single nucleotide polymorphisms ("SNPs”) between individuals.
  • SNPs single nucleotide polymorphisms
  • SNPs include insertions and deletions of simple repeat sequences of varying lengths, such as di- and tri-nucleotide repeats. Accordingly, this application should be understood to extend to any All forms of the marker/gene resulting from other mutations, polymorphisms or allelic variations.
  • the term "marker” shall include both the sense strand sequence of the marker or gene and the antisense strand sequence of the marker or gene.
  • the term "marker” as used herein is to be interpreted broadly to include both 1) the original marker (in a specific methylation state) found in a biological sample or genomic DNA, and 2) its processed sequence (e.g. The corresponding area after bisulfite conversion or the corresponding area after MSRE treatment).
  • the bisulfite-converted corresponding region differs from the target marker in the genomic sequence in that one or more unmethylated cytosine residues are converted to uracil bases, thymine bases, or during hybridization Other bases that behave differently than cytosine.
  • the MSRE-treated corresponding region differs from the target marker in the genomic sequence in that the sequence is cleaved at one or more MSRE cleavage sites.
  • methylation state refers to the presence, absence and/or amount of one or more methylated nucleotide bases in a nucleic acid molecule.
  • a nucleic acid molecule containing methylated cytosine is considered methylated, and the methylation status of the nucleic acid molecule is methylated.
  • Nucleic acid molecules that do not contain any methylated cytosine are considered unmethylated, and the methylation status of the nucleic acid molecule is unmethylated.
  • a nucleic acid may be characterized as "unmethylated” if it is not methylated at a particular locus (eg, the locus of a particular single CpG dinucleotide) or a particular combination of loci, even if It is methylated at other loci in the same gene or molecule.
  • methylation status describes the state of methylation of a nucleic acid (eg, a genomic sequence). Additionally, methylation status refers to the methylation-related characteristics of a nucleic acid segment at a specific genomic locus. Such characteristics include, but are not limited to, whether any cytosine (C) residue within the DNA sequence is methylated, the location of one or more methylated C residues, methylation throughout any specific region of the nucleic acid Frequency or percentage of C and allelic differences in methylation due to, for example, differences in allelic origins. "Methylation status" refers to the relative concentration, absolute concentration, or pattern of methylated C or unmethylated C throughout any particular region of nucleic acid in a biological sample.
  • C cytosine
  • nucleic acid sequence A pyrimidine (C) residue is methylated, then it may be said to be “hypermethylated” or has "increased methylation," whereas if one or more cytosine (C) residues within the DNA sequence is unmethylated, it may be said to be “demethylated” or have “reduced methylation”.
  • cytosine (C) residues within a nucleic acid sequence are methylated compared to another nucleic acid sequence (e.g. from a different region or from a different individual, etc.), the sequence is considered to be different from the other nucleic acid sequence.
  • the nucleic acid sequence is hypermethylated or has increased methylation.
  • cytosine (C) residues within a DNA sequence are unmethylated compared to another nucleic acid sequence (e.g. from a different region or from a different individual, etc.), the sequence is considered to be different from the other.
  • the nucleic acid sequence is demethylated or has reduced methylation.
  • methylation level represents the proportion of one or more sites in a methylated state.
  • the methylation level of a region (or a group of sites) is the average of the methyl levels of all sites in the region (or of all sites in the group). Therefore, an increase or decrease in methylation levels in a region does not mean an increase or decrease in methylation levels at all methylation sites in the region.
  • the process of converting results obtained by methods for detecting DNA methylation eg, simplified methylation sequencing, fluorescence quantitative PCR
  • Methodylation level includes the relationship between the methylation status of any number and any position of CpGs in the sequence involved. The relationship may be the addition or subtraction of methylation state parameters (e.g., 0 or 1) or the calculation result of a mathematical algorithm (e.g., mean, percentage, fraction, ratio, degree, or calculation using a mathematical model), including but not limited to Methylation level measures, methylation haplotype ratios, methylation haplotype loads, or in the case of using machine learning models such as logistic regression models, the AUC of the ROC curve.
  • methylation state parameters e.g., 0 or 1
  • a mathematical algorithm e.g., mean, percentage, fraction, ratio, degree, or calculation using a mathematical model
  • Genes used as markers in the present invention are intended to include naturally occurring variants of the gene, its complementary sequence, all its promoters and regulatory elements (e.g. 5 kb (e.g. 4 kb, 3 kb, 2 kb) upstream of the gene annotation start site or 1 kb) and the nucleic acid sequence within 5 kb downstream of its gene annotation termination site) and fragments of the gene or the variant, especially fragments detectable in molecular biology.
  • the terms "molecularly biologically detectable fragment", “target region” and “target gene region” are used interchangeably.
  • Molecularly biologically detectable fragments preferably comprise at least 16, 17, 18, 19, 20, 22, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 or more consecutive nucleotides.
  • the contiguous nucleotides comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or More CpG dinucleotide sequences.
  • it is preferred that the target gene region is rich in CpG dinucleotides.
  • target region refers to the marker gene itself, 5kb upstream of its gene annotation start site (for example, 4kb, 3kb, 2kb or 1kb) and its gene annotation end site.
  • the target gene region of the target markers in Table 1A, Table 1B and Table 1C below includes its Hg19 coordinate and any molecular biology detectable within 5kb upstream and downstream of the coordinate (such as 4kb, 3kb, 2kb or 1kb) Fragments, their complements or processed sequences (e.g., bisulfite-converted corresponding sequences or MSRE-processed corresponding sequences), and processed sequences (e.g., bisulfite-converted corresponding sequences) of said complementary sequences sequence or the corresponding sequence after MSRE processing).
  • any molecular biology detectable within 5kb upstream and downstream of the coordinate such as 4kb, 3kb, 2kb or 1kb
  • Fragments their complements or processed sequences (e.g., bisulfite-converted corresponding sequences or MSRE-processed corresponding sequences), and processed sequences (e.g., bisulfite-converted corresponding sequences) of said complementary sequences sequence or the corresponding sequence
  • the target gene region of the target marker in Table 1A, Table 1B and Table 1C below includes its Hg19 coordinate and any molecular biology detectable within 5kb (such as 4kb, 3kb, 2kb or 1kb) upstream of the coordinate.
  • fragments, their complementary sequences or processed sequences e.g., bisulfite-converted corresponding sequences or MSRE-processed corresponding sequences
  • processed sequences of the complementary sequences e.g., bisulfite-converted corresponding sequences
  • Corresponding sequence or corresponding sequence after MSRE processing corresponding sequence or corresponding sequence after MSRE processing.
  • target markers and their target gene regions selected from Table 1A, Table 1B and Table 1C below, or any combination thereof:
  • Target markers and target gene regions are listed in Table 1C.
  • TTLL10 described in this article is a protein-coding gene, also known as Tubulin Tyrosine Ligase Like 10, etc.
  • the function of the encoded protein is related to protein glycine ligase activity.
  • the EPS8L3 described in this article is a protein-coding gene, also known as EPS8Like 3, etc.
  • the encoded protein is related to epidermal growth factor receptor pathway substrate 8.
  • the IRF2BP2 described in this article is a protein-coding gene, also known as Interferon Regulatory Factor 2 Binding Protein 2, etc.
  • the encoded protein combines with the IRF2 protein to form a transcription repression complex.
  • the FAM150B described in this article is a protein-coding gene, also known as ALKAL2, ALK And LTK Ligand 2, etc.
  • the encoded protein is the ligand of the tyrosine kinase ALK and LTK receptors.
  • the ID2 described in this article is a protein-coding gene, also known as Inhibitor Of DNA Binding 2, etc.
  • the function of the encoded protein is related to transcriptional regulation.
  • TERT described in this article is a protein-coding gene, also known as Telomerase Reverse Transcriptase, etc.
  • the encoded protein is involved in the apoptosis of synovial fibroblasts and the WNT signaling pathway.
  • PITX1 described in this article is a protein-coding gene, also known as Paired Like Homeodomain 1, etc., which functions as a transcriptional regulator to activate gene expression.
  • KCNMB1 described in this article is a protein-coding gene, also known as Potassium Calcium-Activated Channel Subfamily M Regulatory Beta Subunit 1.
  • the encoded protein is related to the activity of potassium and calcium ion channels.
  • BEND6 described in this article is a protein-coding gene, also known as BEN Domain Containing 6, encoding a protein involved in the Notch signaling pathway.
  • the ELN described in this article is a protein-coding gene, also known as Elastin, which encodes proteins involved in forming the extracellular matrix.
  • CPXM2 described in this article is a protein-coding gene, also known as Carboxypeptidase X, M14 Family Member 2.
  • the encoded protein is related to metallocarboxypeptidase activity.
  • TH described in this article is a protein-coding gene, also known as Tyrosine Hydroxylase.
  • the encoded protein functions as tyrosine hydroxylase.
  • the C1QTNF9 described in this article is a protein-coding gene, also known as C1q And TNF Related 9.
  • the encoded protein activates AMPK, AKT, and p44/42MAPK signaling pathways.
  • the CARKD described in this article is a protein-coding gene, also known as NAXD and NAD(P)HX Dehydratase.
  • the encoded protein is involved in the metabolism of water-soluble vitamins and cofactors and the niacin metabolism pathway.
  • TMEM179 described in this article is a protein-coding gene, also known as Transmembrane Protein 179.
  • SPNS1 described in this article is a protein-coding gene, also known as Sphingolipid Transporter 1 (Putative).
  • the function of the coding protein is related to the activity of the transporter.
  • MYO15B described in this article is a protein-coding gene, also known as Myosin XVB.
  • DNM2 described in this article is a protein-coding gene, also known as Dynamin 2.
  • the encoded protein is one of the subclasses of GTP-binding proteins.
  • the EPHX3 described in this article is a protein-coding gene, also known as Epoxide Hydrolase 3, which encodes a protein that catalyzes the hydrolysis of epoxy-containing fatty acids.
  • the PSG8 described in this article is a protein-coding gene, also known as Pregnancy Specific Beta-1-Glycoprotein 8.
  • the encoded protein participates in the blood vessel wall cell surface interaction signaling pathway and the platelet calcium ion increase response signaling pathway.
  • SLCO4A1 described in this article is a protein-coding gene, also known as Solute Carrier Organic Anion Transporter Family Member 4A1.
  • the encoded protein is related to transport activity.
  • TNFRSF6B described in this article is a protein-coding gene, also known as TNF Receptor Superfamily Member 6b.
  • the encoded protein belongs to the tumor necrosis factor receptor family.
  • the target marker CARKD and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • TTLL10 TTLL10
  • EPS8L3, IRF2BP2 or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • TTLL10 and its target gene region it is preferred to use and detect the target marker TTLL10 and its target gene region, optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1, and TNFRSF6B.
  • TTLL10 TTLL10
  • EPS8L3, IRF2BP2 any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO
  • the target marker EPS8L3 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: TTLL10, IRF2BP2, FAM150B , ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • a target marker and its target gene region selected from the following: TTLL10, IRF2BP2, FAM150B , ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • the target marker IRF2BP2 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: TTLL10, EPS8L3, FAM150B , ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • a target marker and its target gene region selected from the following: TTLL10, EPS8L3, FAM150B , ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • the target marker FAM150B and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: TTLL10, EPS8L3, IRF2BP2, or any combination thereof ,ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • TTLL10 TTLL10
  • EPS8L3, IRF2BP2 or any combination thereof ,ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • a target marker and its target gene region selected from the group consisting of: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • the target marker TERT and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • TTLL10 TTLL10
  • EPS8L3, IRF2BP2 or any combination thereof , FAM150B, ID2, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • the target marker PITX1 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • the target marker KCNMB1 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • target marker BEND6 and its target base Because of the region, optionally additionally use and detect target markers and their target gene regions selected from the following: TTLL10, EPS8L3, IRF2BP2, FAM150B, ID2, TERT, PITX1, KCNMB1, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • target markers and their target gene regions selected from the following: TTLL10, EPS8L3, IRF2BP2, FAM150B, ID2, TERT, PITX1, KCNMB1, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • a target marker and its target gene region selected from the group consisting of: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • the target marker CPXM2 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • a target marker and its target gene region selected from the group consisting of: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • the target marker C1QTNF9 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • a target marker and its target gene region selected from the following: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, SPNS1, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • the target marker SPNS1 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • TTLL10 TTLL10
  • EPS8L3, IRF2BP2 or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, MYO15B, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • the target marker MYO15B and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • TTLL10 TTLL10
  • EPS8L3, IRF2BP2 or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, DNM2, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • the target marker DNM2 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, EPHX3, PSG8, SLCO4A1 and TNFRSF6B.
  • the target marker EPHX3 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: TTLL10, EPS8L3, IRF2BP2, or any combination thereof ,FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, PSG8, SLCO4A1 and TNFRSF6B.
  • the target marker PSG8 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, SLCO4A 1 and TNFRSF6B.
  • TTLL10 TTLL10
  • EPS8L3, IRF2BP2 or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, SLCO4A 1 and TNFRSF6B.
  • the target marker SLCO4A1 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8 and TNFRSF6B.
  • TTLL10 TTLL10
  • EPS8L3, IRF2BP2 or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8 and TNFRSF6B.
  • a target marker and its target gene region selected from the following: TTLL10, EPS8L3, IRF2BP2, or any combination thereof , FAM150B, ID2, TERT, PITX1, KCNMB1, BEND6, ELN, CPXM2, TH, C1QTNF9, CARKD, TMEM179, SPNS1, MYO15B, DNM2, EPHX3, PSG8 and SLCO4A1.
  • the SKI described in this article is a protein-coding gene, also known as SKI Proto-Oncogene.
  • the encoded protein functions as an inhibitor of the TGF-beta signaling pathway.
  • PRDM16 described in this article is a protein-coding gene, also known as PR/SET Domain 16.
  • the encoded protein can regulate gene transcription.
  • PIAS3 described in this article is a protein-coding gene, also known as Protein Inhibitor Of Activated STAT 3.
  • the encoded protein inhibits the activation of the STAT3 signaling pathway.
  • the SLC10A4 described in this article is a protein-coding gene, also known as Solute Carrier Family 10 Member 4.
  • the encoded protein is a transporter that participates in the transport of bile acids and other substances.
  • the CXXC5 described in this article is a protein-coding gene, also known as CXXC Finger Protein 5.
  • the encoded protein binds to specific DNA motifs and participates in signal transduction of multiple signaling pathways such as NF-kappa-B, MAPK, and WNT.
  • the NR2E1 described in this article is a protein-coding gene, also known as Nuclear Receptor Subfamily 2 Group E Member 1.
  • the coding protein participates in the formation of nuclear receptors.
  • MPC1 described in this article is a protein-coding gene, also known as Mitochondrial Pyruvate Carrier1, encoding a protein involved in mitochondrial pyruvate transport.
  • HOXA13 described in this article is a protein-coding gene, also known as Homeobox A13.
  • the encoded protein is a transcription factor and participates in transcriptional regulation.
  • the LZTS1 described in this article is a protein-coding gene, also known as Leucine Zipper Tumor Suppressor 1.
  • the encoded protein is involved in regulating the cell cycle.
  • CHD7 described in this article is a protein-coding gene, also known as Chromodomain Helicase DNA Binding Protein 7.
  • the participating gene ontology annotations are chromatin binding and helicase activities.
  • ANKRD20A1 described in this article is a protein-coding gene, also known as Ankyrin Repeat Domain 20 Family Member A1.
  • the CACNA1B described in this article is a protein-coding gene, also known as Calcium Voltage-Gated Channel Subunit Alpha1 B.
  • the encoded protein participates in the formation of calcium ion channels.
  • ACVRL1 described in this article is a protein-coding gene, also known as Activin A Receptor Like Type 1.
  • the encoded protein is a TFG-beta family ligand receptor, which is involved in regulating blood vessel development.
  • CCNA1 described in this article is a protein-coding gene, also known as Cyclin A1.
  • the encoded protein is involved in regulating the cell cycle.
  • RNASEH2B described in this article is a protein-coding gene, also known as Ribonuclease H2 Subunit B.
  • the encoded protein is an endonuclease subunit.
  • SNX20 described in this article is a protein-coding gene, also known as Sorting Nexin 20, edited by Encoded proteins are involved in cellular vesicle transport.
  • the TBCD described in this article is a protein-coding gene, also known as Tubulin Folding Cofactor D, which encodes a protein involved in tubulin folding.
  • the PIP5K1C described in this article is a protein-coding gene, also known as Phosphatidylinositol-4-Phosphate 5-Kinase Type 1 Gamma, which encodes a protein that functions as a phosphokinase.
  • ZBTB7A described in this article is a protein-coding gene, also known as Zinc Finger And BTB Domain Containing 7A.
  • the encoded protein is a transcription factor and inhibits transcriptional activation.
  • DNASE2 described in this article is a protein-coding gene, also known as Deoxyribonuclease2, Lysosomal.
  • the encoded protein belongs to the DNA endonuclease family.
  • TSHZ3 described in this article is a protein-coding gene, also known as Teashin Zinc Finger Homeobox 3.
  • the encoded protein is involved in transcriptional regulation.
  • WISP2 described in this article is a protein-coding gene, also known as CCN5 or Cellular Communication Network Factor 5.
  • the encoded protein is a member of the WISP protein family.
  • the target marker SKI and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: PRDM16, PIAS3, SLC10A4, or any combination thereof , CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • a target marker and its target gene region selected from the group consisting of: PRDM16, PIAS3, SLC10A4, or any combination thereof , CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A,
  • target marker PRDM16 and its target gene region optionally additionally using and detecting target markers and their target genes selected from: regions or any combination thereof: SKI, PIAS3, SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3, and WISP2.
  • target markers and their target genes selected from: regions or any combination thereof: SKI, PIAS3, SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3, and WISP2.
  • the target marker PIAS3 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, SLC10A4, or any combination thereof , CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, SLC10A4, or any combination thereof , CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A,
  • the target marker SLC10A4 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, PIAS3 or any combination thereof , CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • the target marker CXXC5 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, PIAS3, or any combination thereof , SLC10A4, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • the target marker NR2E1 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, PIAS3, or any combination thereof , SLC10A4, CXXC5, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, PIAS3, or any combination thereof , SLC10A4, CXXC5, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A,
  • the target marker MPC1 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSH
  • the target marker HOXA13 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, PIAS3, or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, PIAS3, or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2,
  • the target marker LZTS1 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ
  • the target marker CHD7 and its target gene region are preferably used and detected, optionally additionally selected from the following target markers and their target gene region or any combination thereof: SKI, PRDM16, PIAS3 , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • the target marker ANKRD20A1 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • the target marker CACNA1B and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, PIAS3, or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS 1, CHD7, ANKRD20A1, ACVRL 1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • the target marker ACVRL1 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, PIAS3, or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS 1, CHD7, ANKRD20A1, CACNA1B, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, PIAS3, or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS 1, CHD7, ANKRD20A1, CACNA1B, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A,
  • the target marker CCNA1 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ
  • RNASEH2B and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • the target marker SNX20 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following domains or any combination thereof: SKI, PRDM16, PIAS3, SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3, and WISP2.
  • a target marker and its target gene region selected from the following domains or any combination thereof: SKI, PRDM16, PIAS3, SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, TBCD, PIP5K1C, ZBTB7A, DNASE2, TSHZ3,
  • the target marker TBCD, and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3, SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS 1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3, SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS 1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, PIP5K1C, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • the target marker PIP5K1C and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS 1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, ZBTB7A, DNASE2, TSHZ3 and WISP2.
  • a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS 1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, ZBTB7A, DNASE2, TSHZ3 and WISP
  • the target marker ZBTB7A and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS 1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, DNASE2, TSHZ3 and WISP2.
  • a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS 1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, DNASE2, TSHZ3 and WI
  • the target marker DNASE2 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS 1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, TSHZ3 and WISP2.
  • a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS 1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, TSH
  • the target marker TSHZ3 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, PIAS3, or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2, and WISP2.
  • a target marker and its target gene region selected from the group consisting of: SKI, PRDM16, PIAS3, or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBT
  • the target marker WISP2 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE2 and TSHZ3.
  • a target marker and its target gene region selected from the following: SKI, PRDM16, PIAS3 or any combination thereof , SLC10A4, CXXC5, NR2E1, MPC1, HOXA13, LZTS1, CHD7, ANKRD20A1, CACNA1B, ACVRL1, CCNA1, RNASEH2B, SNX20, TBCD, PIP5K1C, ZBTB7A, DNASE
  • WRAP73 described in this article is a protein-coding gene, also known as WD Repeat Containing, Antisense To TP73, and the encoded protein is a member of the WD repeat protein family.
  • C2CD4D described in this article is a protein-coding gene, also known as C2 Calcium Dependent Domain Containing 4D.
  • CCDC181 described in this article is a protein-coding gene, also known as Coiled-Coil Domain Containing 181.
  • RNF144A described in this article is a protein-coding gene, also known as Ring Finger Protein 144A.
  • the encoded protein belongs to the zinc finger protein family.
  • SIX2 described in this article is a protein-coding gene, also known as SIX Homeobox 2, and the encoded protein is a transcription factor.
  • NRXN1 described in this article is a protein-coding gene, also known as Neurexin 1, and the encoded protein is a cell surface protein.
  • MEIS1 described in this article is a protein-coding gene, also known as Meis Homeobox 1, and the encoded protein is the transcriptional regulator of PAX6.
  • LBX2 described in this article is a protein-coding gene, also known as Ladybird Homeobox 2, and the encoded protein is a transcriptional regulatory factor.
  • the AMT described in this article is a protein-coding gene, also known as Aminomethyltransferase, encoding a protein involved in glycine degradation.
  • the ITIH4 described in this article is a protein-coding gene, also known as Inter-Alpha-Trypsin Inhibitor Heavy Chain 4, and the encoded protein is a secreted protein.
  • TRH described in this article is a protein-coding gene, also known as Thyrotropin Releasing Hormone.
  • the encoded protein is a member of the thyrotropin-releasing hormone family.
  • SHOX2 described in this article is a protein-coding gene, also known as Short Stature Homeobox 2, and the encoded protein is a transcription factor.
  • the DGKG described in this article is a protein-coding gene, also known as Diacylglycerol Kinase Gamma.
  • the encoded protein belongs to the diacylglycerol kinase family.
  • the RPL9 described in this article is a protein-coding gene, also known as Ribosomal Protein L9, which encodes proteins involved in constituting ribosome subunits.
  • PFN3 described in this article is a protein-coding gene, also known as Profilin 3.
  • the encoded protein belongs to the actin family.
  • FOXC1 described in this article is a protein-coding gene, also known as Forkhead Box C1, and the encoded protein is a transcription factor.
  • LY86 described in this article is a protein-coding gene, also known as Lymphocyte Antigen 86.
  • the encoded protein participates in immune-related signaling pathways.
  • the SLC35F1 described in this article is a protein-coding gene, also known as Solute Carrier Family 35Member F1.
  • the encoded protein is a member of the ion transport family.
  • the LRRC4 described in this article is a protein-coding gene, also known as Leucine Rich Repeat Containing 4, and the encoded protein is synaptic adhesion protein.
  • PDLIM2 described in this article is a protein-coding gene, also known as PDZ And LIM Domain 2.
  • the coding protein function is related to cell adhesion.
  • PAX2 described in this article is a protein-coding gene, also known as Paired Box 2, and the encoded protein is a transcription factor.
  • the MVK described in this article is a protein-coding gene, also known as Mevalonate Kinase, and the encoded protein is mevalonate kinase.
  • the DTX1 described in this article is a protein-coding gene, also known as Deltex E3 Ubiquitin Ligase 1.
  • the encoded protein is a ubiquitination ligase.
  • RBM19 described in this article is a protein-coding gene, also known as RNA Binding Motif Protein 19.
  • the GCH1 described in this article is a protein-coding gene, also known as GTP Cyclohydrolase 1.
  • the encoded protein is a member of the GTP cyclohydrolase family.
  • the OTX2 described in this article is a protein-coding gene, also known as Orthodenticle Homeobox 2, and the encoded protein is a transcription factor.
  • ZSCAN10 described in this article is a protein-coding gene, also known as Zinc Finger And SCAN Domain Containing 10.
  • the encoded protein is a transcription factor.
  • the AHSP described in this article is a protein-coding gene, also known as Alpha Hemoglobin Stabilizing Protein.
  • the NLRC5 described in this article is a protein-coding gene, also known as NLR Family CARD Domain Containing 5.
  • the encoded protein is involved in regulating the immune system.
  • the ASXL3 described in this article is a protein-coding gene, also known as ASXL Transcriptional Regulator 3.
  • the encoded protein is involved in regulating gene transcription.
  • TCF4 described in this article is a protein-coding gene, also known as Transcription Factor 4, and the encoded protein is a transcription factor.
  • PLIN3 described in this article is a protein-coding gene, also known as Perilipin 3.
  • RASAL3 described in this article is a protein-coding gene, also known as RAS Protein Activator Like 3, and the encoded protein is a member of the RasGAP family.
  • CHRNA4 described in this article is a protein-coding gene, also known as Cholinergic Receptor Nicotinic Alpha 4 Subunit.
  • the encoded protein is the nicotinic acetylcholine receptor subunit.
  • the target marker WRAP73 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: C2CD4D, CCDC181, RNF144A , SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: C2CD4D, CCDC181, RNF144A , SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4,
  • the target marker C2CD4D and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, CCDC181, RNF144A , SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, CCDC181, RNF144A , SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, P
  • the target marker CCDC181 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, RNF144A , SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, RNF144A , SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LR
  • the target marker RNF144A and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 ,SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5, ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 ,SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LR
  • the target marker SIX2 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • WRAP73 WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, Z
  • the target marker MEIS1 and its target gene region are preferably used and detected, optionally additionally selected from the following target markers and their target gene region or any combination thereof: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • the target marker LBX2 and its target gene region optionally additionally using and detecting target markers and its target gene region selected from the following Domains or any combination thereof: WRAP73, C2CD4D, CCDC181, RNF144A, SIX2, NRXN1, MEIS1, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1 , RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5, ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • Target markers and its target gene region selected from the following Domains or any combination thereof: WRAP73, C2CD4D, CCDC181, RNF144A, SIX2, NRXN1, MEIS1, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, S
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • WRAP73 WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1,
  • the target marker ITIH4 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1,
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • WRAP73 WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1,
  • target marker SHOX2 and its target base Because of the region, optionally additionally use and detect target markers and their target gene regions selected from the following: WRAP73, C2CD4D, CCDC181, RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5, ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • target markers and their target gene regions selected from the following: WRAP73, C2CD4D, CCDC181, RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2,
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • WRAP73 WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX
  • the target marker RPL9 and its target gene region are preferably used and detected, optionally additionally selected from the following target markers and their target gene region or any combination thereof: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • the target marker PFN3 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, FOXC1, LY86, SLC35F
  • target markers and their target gene regions selected from the following: WRAP73, C2CD4D, CCDC181, RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5, ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • target markers and their target gene regions selected from the following: WRAP73, C2CD4D, CCDC181, RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX
  • the target marker LY86 and its target gene region are preferably used and detected, optionally additionally selected from the following target markers and their target gene region or any combination thereof: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • the target marker SLC35F1 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86,
  • the target marker LRRC4 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, S
  • the target marker PDLIM2 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, S
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • WRAP73 WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, MVK, DTX1, RBM19, GCH1,
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • WRAP73 WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, DTX1, RBM19, GCH1, OTX
  • the target marker DTX1 and its target gene region are preferably used and detected, optionally additionally selected from the following target markers and their target gene region or any combination thereof: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5, ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • the target marker RBM19 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, GCH1, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC
  • the target marker GCH1 and its target gene region are preferably used and detected, optionally additionally selected from the following target markers and their target gene region or any combination thereof: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, OTX2, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • the target marker OTX2 and its target gene region are preferably used and detected, optionally additionally selected from the following target markers and their target gene region or any combination thereof: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, ZSCAN10, AHSP, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • the target marker ZSCAN10 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, AHSP, NLRC5, ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, NLRC5 , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • WRAP73 WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP , ASXL3, TCF4, PLIN3, RASAL3 and CHRNA4.
  • WRAP73 WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1,
  • the target marker ASXL3 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP , NLRC5, TCF4, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC
  • the target marker TCF4 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP, NLRC5, ASXL3, PLIN3, RASAL3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35
  • the target marker PLIN3 and its target gene region are preferably used and detected, optionally additionally selected from the following target markers and their target gene region or any combination thereof: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP , NLRC5, ASXL3, TCF4, RASAL3 and CHRNA4.
  • the target marker RASAL3 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP , NLRC5, ASXL3, TCF4, PLIN3 and CHRNA4.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC
  • the target marker CHRNA4 and its target gene region optionally additionally using and detecting a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC35F1, LRRC4, PDLIM2, PAX2, MVK, DTX1, RBM19, GCH1, OTX2, ZSCAN10, AHSP , NLRC5, ASXL3, TCF4, PLIN3 and RASAL3.
  • a target marker and its target gene region selected from the following: WRAP73, C2CD4D, CCDC181 , RNF144A, SIX2, NRXN1, MEIS1, LBX2, AMT, ITIH4, TRH, SHOX2, DGKG, RPL9, PFN3, FOXC1, LY86, SLC
  • At least 25% can be achieved with a specificity of greater than 80%, such as greater than 85% or greater than 90%, using the target markers of the invention and their target gene regions or combinations thereof.
  • subject refers to warm-blooded animals, such as mammals.
  • the term includes, but is not limited to, domestic animals, rodents (eg, rats and mice), primates, and humans. Preferably the term refers to humans.
  • methylation assay refers to any assay that determines the methylation status of one or more dinucleotide (eg, CpG) sequences within a DNA sequence.
  • the term "threshold” should be understood according to the general understanding of those skilled in the art, and means any useful reference for reflecting DNA methylation levels.
  • the threshold is expressed as a positive reference interval, wherein within the positive reference interval indicates that the individual has breast cancer or is at risk for breast cancer; e.g., one or more markers compared to the corresponding positive reference interval A methylation level within the positive reference interval indicates that the individual has breast cancer or is at risk for breast cancer.
  • Thresholds or positive reference intervals can be obtained from known databases or from individual studies.
  • threshold or positive reference interval refers to the level from a positive control (ie, an individual with breast cancer).
  • the threshold or positive reference interval may be obtained from a reference sample of the patient's own blood; expression of the marker gene from an individual with breast cancer; or predetermined breast cancer cells from an individual with breast cancer.
  • the AUC value of the ROC curve is used to set a positive reference interval, such as greater than 90% specificity for each marker (not The AUC value (the proportion of breast cancer samples testing positive is less than 10%) sets the positive reference interval.
  • the AUC threshold is set to, for example, be equal to or greater than 0.362.
  • the AUC threshold is set, for example, to be equal to or greater than 0.609.
  • the AUC threshold is set, for example, to be equal to or greater than 0.604.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.534.
  • the AUC threshold is set, for example, to be equal to or greater than 0.531.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.534.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.534.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.537.
  • the AUC threshold is set, for example, to be equal to or greater than 0.531.
  • the AUC value of the ROC curve is used to set a positive reference interval, such as greater than 90% specificity for each marker (not The AUC value (the proportion of breast cancer samples testing positive is less than 10%) sets the positive reference interval.
  • the AUC threshold is set to, for example, be equal to or greater than 0.440.
  • the AUC threshold is set, for example, to be equal to or greater than 0.541.
  • the AUC threshold is set, for example, to be equal to or greater than 0.513.
  • the AUC threshold is set, for example, to be equal to or greater than 0.534.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.534.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.540.
  • the AUC threshold is set, for example, to be equal to or greater than 0.534.
  • the AUC threshold is set, for example, to be equal to or greater than 0.534.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.530.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.530.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.548.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.529.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC value of the ROC curve is used to set a positive reference interval, such as greater than 90% specificity for each marker (not The AUC value (the proportion of breast cancer samples testing positive is less than 10%) sets the positive reference interval.
  • the AUC threshold is set to, for example, be equal to or greater than 0.505.
  • the AUC threshold is set, for example, to be equal to or greater than 0.445.
  • the AUC threshold is set, for example, to be equal to or greater than 0.447.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.534.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.529.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.534.
  • the AUC threshold is set, for example, to be equal to or greater than 0.531.
  • the AUC threshold is set, for example, to be equal to or greater than 0.535.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.535.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.531.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • the AUC threshold is set, for example, to be equal to or greater than 0.531.
  • the AUC threshold is set, for example, to be equal to or greater than 0.533.
  • the AUC threshold is set, for example, to be equal to or greater than 0.532.
  • oligonucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The term includes double-stranded and single-stranded DNA and RNA, modified forms such as methylation or capping, and unmodified forms of the polynucleotide.
  • polynucleotide and “oligonucleotide” are used interchangeably herein.
  • An oligonucleotide may, but need not, include other coding or non-coding sequences, or it may, but need not, be linked to other molecules and/or vectors or support materials.
  • Oligonucleotides used in the methods or kits of the invention can be of any length suitable for the particular method.
  • the term refers to an antisense nucleic acid molecule (eg, an mRNA or DNA strand in the opposite orientation to the sense polynucleotide encoding the marker of the invention).
  • Oligonucleotides useful in the present invention include complementary nucleic acid sequences and nucleic acids that are substantially identical to these sequences, and also include sequences that differ from the nucleic acid sequences due to degeneracy of the genetic code. Oligonucleotides useful in the present invention also include nucleic acids that hybridize to the oligonucleotide cancer marker nucleic acid sequence under stringent conditions, preferably under high stringency conditions.
  • Nucleotide hybridization assays are well known in the art. Hybridization assay procedures and conditions will vary depending on the application and are chosen based on known general binding methods, see e.g. J. Samburu Gram et al., Molecular Cloning: A Laboratory Guide (3rd ed. Science Press, 2002); and Young and Davis, PNAS, 80: 1194 (1983). Methods and equipment for performing repetitive and controlled hybridization reactions have been described in U.S. Patent Nos. 5,871,928, 5,874,219, 6,045,996, 6,386,749, and 6,391,623, each of which is incorporated herein by reference.
  • primer generally refers to a linear oligonucleotide that is complementary to and anneals to a target sequence.
  • the lower limit of primer length is determined by hybridization ability because very short primers (eg, less than 5 nucleotides) do not form thermodynamically stable duplexes under most hybridization conditions.
  • Primer length typically varies from 8-50 nucleotides.
  • primers are between about 15-25 nucleotides.
  • Naturally occurring nucleotides especially guanine, adenine, cytosine and thymine, hereafter referred to as "G", "A”, "C” and “T”
  • G Naturally occurring nucleotides
  • amplification product refers to amplified nucleic acid produced by nucleic acid amplification from a nucleic acid template.
  • nucleotide analog refers to compounds that are structurally similar to naturally occurring nucleotides. Nucleotide analogs may have altered phosphate backbones, sugar moieties, nucleobases, or combinations thereof. Nucleotide analogs, often with altered nucleobases, confer inter alia different base pairing and base stacking properties. Nucleotide analogs with an altered phosphate-sugar backbone (eg, peptide nucleic acids (PNA), locked nucleic acids (LNA)) often alter chain properties, such as secondary structure formation, among others.
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • primers and probes used in the present invention are shown in Table 2A, Table 2B and Table 2C, and the target gene regions they target are shown in Table 1A, Table 1B and Table 1C.
  • R represents random base A, T, C or G.
  • R represents random base A, T, C or G.
  • R represents random base A, T, C or G.
  • the nucleotide sequences of the primers and probes of the present invention also include modified forms thereof, as long as the amplification or detection effects of the primers are not significantly affected.
  • the modification may be, for example, the addition of one or more nucleotide residues in or at both ends of the nucleotide sequence, the deletion of one or more nucleotide residues in the nucleotide sequence, or the addition of one or more nucleotide residues in the sequence. Multiple nucleotide residues are replaced with additional nucleotide residues, for example, A is replaced by T, C is replaced by G, etc. It is clear to those skilled in the art that the modified primers are also covered by the present invention, especially within the protection scope of the claims.
  • the modified form of the nucleotide sequence of the primer is a chemically enhanced primer as disclosed in CN103270174A.
  • Each nucleotide in the primer of the invention can be chemically synthesized using, for example, a general-purpose DNA synthesizer (eg, Model 394 manufactured by Applied Biosystems). Oligonucleotides may also be synthesized using any other method well known in the art.
  • a general-purpose DNA synthesizer eg, Model 394 manufactured by Applied Biosystems.
  • Oligonucleotides may also be synthesized using any other method well known in the art.
  • Amplification reactions include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCP), automatically maintained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), and strand displacement amplification (SDA). , multiple displacement amplification (MDA) and rolling circle amplification (RCA), which are disclosed in the following references (herein incorporated by reference): Mullis et al., U.S. Patent No. 4,683,195; No. 4,965,188; No. 4,683,202; No. 4,800,159 (PCR); Gelfand et al., U.S. Patent No.
  • Target markers are preferably amplified using PCR.
  • the PCR method itself is well known in the art.
  • the term "PCR” includes derivative forms of this reaction, including but not limited to reverse transcription PCR, real-time PCR, nested PCR, multiplex PCR, fluorescence quantitative PCR, and the like. It is preferable to use fluorescence quantitative PCR method to quantitatively amplify the target nucleotide.
  • PCR uses a primer that hybridizes to the sense strand (reverse primer) and a primer that hybridizes to the antisense strand (forward primer) in the presence of primers, template DNA, and thermostable DNA polymerase.
  • reverse primer a primer that hybridizes to the sense strand
  • forward primer a primer that hybridizes to the antisense strand
  • the steps of denaturation, annealing, and extension are PCR is performed by repeating the cycle approximately 30 to 60 times (for example, 50 times).
  • the PCR is fluorescence quantitative PCR.
  • PCR uses primers as shown in Table 2. Those skilled in the art can understand that other PCR methods and primers can also be used, as long as the target fragment can be amplified.
  • thermostable DNA polymerases can be used for amplification, including but not limited to FastStart Taq DNA polymerase (Roche), Ex Taq (registered trademark, Takara), Z-Taq, AccuPrime Taq DNA polymerase and HotStarTaq Plus DNA polymerase.
  • the method of selecting appropriate PCR reaction conditions based on primer Tm values is well known in the art. Those of ordinary skill in the art can select the optimal conditions based on primer length, GC content, target specificity and sensitivity, properties of the polymerase used, etc. For example, the following conditions can be used for fluorescence quantitative PCR reaction: 95°C for 5 minutes; 95°C for 15 seconds, 56°C for 40 seconds, and 50 cycles.
  • the reaction system is 25 ⁇ L.
  • Reagents useful for detecting methylation levels of target markers of the invention are well known in the art.
  • Such reagents suitable for use in the present invention such as bisulfite reagents or methylation-sensitive restriction enzymes, are commercially available or are routinely prepared by methods well known to those skilled in the art.
  • bisulfite reagent refers to bisulfite used to distinguish between methylated and unmethylated CpG dinucleotide sequences.
  • methylation-sensitive restriction enzyme is understood to mean an enzyme that selectively digests nucleic acids depending on the methylation status of its recognition site. For restriction enzymes that specifically cleave when the recognition site is unmethylated or hemimethylated, when the recognition site is methylated, cleavage does not occur or cleaves with significantly reduced efficiency. cut. For restriction enzymes that specifically cleave only when the recognition site is methylated, when the recognition site is not methylated, cleavage will not occur, or it will cleave with significantly reduced efficiency. Preferred are methylation-sensitive restriction enzymes whose recognition sequences contain CG dinucleotides (eg cgcg or cccggg). In some embodiments Of these, restriction enzymes that do not cleave when cytosine in the dinucleotide is methylated at the C5 carbon atom are further preferred.
  • Kits may contain materials or reagents for performing the methods of the invention (including reagents for detecting each target marker).
  • Kits may include storage of reaction reagents (eg, primers, dNTPs, enzymes, etc. in appropriate containers) and/or supporting materials (eg, buffers, instructions for performing the assay, etc.).
  • a kit may include one or more containers (eg, boxes) containing corresponding reaction reagents and/or support materials. Such contents may be delivered together or separately to the intended recipient.
  • a kit may contain reagents for detecting each target marker, buffers, and instructions for use.
  • the kit may also contain polymerase, dTNP, etc.
  • the kit can also contain internal standards, positive and negative controls for quality control.
  • the kit may also contain reagents for preparing nucleic acids, such as DNA, from the sample.
  • nucleic acids such as DNA
  • a microarray refers to a solid support with a flat surface that contains an array of nucleic acids, each member of the array containing an identical copy of an oligonucleotide or polynucleotide fixed at a spatially defined region or site, so The regions or sites do not overlap with regions or sites of other members of the array; that is, the regions or sites are spatially discrete.
  • a spatially defined hybridization site may be "addressable" in that its location and the identity of its immobilized oligonucleotide are known or predetermined (e.g., prior to its use). definite).
  • oligonucleotide or polynucleotide is single-stranded and is usually covalently linked to a solid support from the 5'-end or 3'-end.
  • the density of nucleic acids containing non-overlapping regions in the microarray is generally greater than 100/em 2 , more preferably greater than 1000/cm 2 .
  • Microarray technology is disclosed, for example, in the following references: Microarrays: A Practical Approach, edited by Schena (IRL Press, Oxford, 2000); Southern, Current Opin. Chem. Biol., 2: 404-410, 1998, the entire contents of which are adopted by This reference is incorporated into this article.
  • the present invention discloses the use of markers in diagnosing breast cancer and predicting its risk. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters. In particular, it should be noted that all similar substitutions and modifications will be obvious to those skilled in the art. It is obvious that they are all considered to be included in the present invention.
  • the uses described in the present invention have been described through preferred embodiments. Relevant persons can obviously make changes or appropriate changes and combinations to the uses described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention. .
  • Example 1 Methylation-targeted sequencing to screen breast cancer methylation markers in plasma
  • This application uses the method of Methyl-Titan (Chinese Patent No. CN201910515830) to obtain methylation sequencing data of sample plasma cfDNA, and screen out the methylation markers.
  • the library is subjected to 150bp paired-end sequencing using an Illumina Nextseq 500 sequencer, and the sequencing volume is no less than 5M.
  • Pear (v0.6.0) software merges the paired-end sequencing data of the same fragment of paired-end 150bp sequencing off the sequencer into one sequence, with a minimum overlap length of 20bp and a minimum length of 30bp after merging.
  • the reference genome data used in this article comes from the UCSC database (UCSC: HG19, http://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/hg19.fa.gz).
  • M is the total number of CpG sites in the target methylation interval
  • i is the CpG site in the interval
  • N C is the number of reads sequenced as C for the CpG site (i.e., methylation reads number)
  • N T is the number of reads sequenced as T for this CpG site (that is, the number of unmethylated sequencing reads).
  • a target methylation interval may have multiple methylation haplotype haplotypes. This value needs to be calculated for each methylation haplotype haplotype in the target area.
  • An example of the MHF calculation formula is as follows:
  • l represents the target methylation interval
  • h represents the target methylation haplotype
  • N l represents the number of reads located in the target methylation interval
  • N l represents the number of reads containing the target methylation haplotype
  • the MHF value is the feature matrix of the training set and test set, and the target methylation interval with the number of reads less than 100 is regarded as a missing value. .
  • a total of 79 breast cancer methylation markers were screened out through the above process (22 in group (1), 22 in group (2), and 35 in group (3)), of which one or more methylation markers were material Both combinations can be used as methylation markers for breast cancer identification.
  • Methylation marker-associated genes refer to the genes corresponding to the nearest TSS within 100Kb from the methylation marker. The specific associated genes and methylation levels are shown in Table 4A.
  • Methylation marker-associated genes refer to the genes corresponding to the nearest TSS within 100Kb of the methylation marker.
  • Seq ID NO: 14 We use Seq ID NO: 14 to show in detail the methylation levels of this methylation marker in the training set and test set of breast cancer and healthy people, as shown in Table 4A below.
  • This methylation marker has high levels in the training set and test set of breast cancer and healthy people. Healthy people have extremely significant methylation differences, with the training set Wilcox.P value being 1.2E-11 and the test set Wilcox.P value being 1.0E-08.
  • Methylation marker-associated genes refer to the genes corresponding to the nearest TSS within 100Kb from the methylation marker. The specific associated genes and methylation levels are shown in Table 4B.
  • Methylation marker-associated genes refer to the genes corresponding to the nearest TSS within 100Kb of the methylation marker.
  • Seq ID NO: 75 We use Seq ID NO: 75 to show in detail the methylation level of this methylation marker in the training set and test set of breast cancer and healthy people, as shown in Table 4B below.
  • This methylation marker has high levels in the training set and test set of breast cancer and healthy people. Healthy people have extremely significant methylation differences, with the training set Wilcox.P value being 9.6E-12 and the test set Wilcox.P value being 2.5E-03.
  • Methylation marker-associated genes refer to the genes corresponding to the nearest TSS within 100Kb of the methylation marker. The specific associated genes and methylation levels are shown in Table 4C.
  • the methylation levels of group (3) 35 methylation markers in the training set and test set of breast cancer samples and healthy human samples are shown in Figure 1 and Table 4C.
  • the genomic location of a methylation marker refers to the location of the methylation marker in the UCS
  • Methylation marker-associated genes refer to the TSS within 100Kb of the methylation marker. And the closest gene. Calculate the methylation marker methylation level of each sample for the training set and test set of healthy and breast cancer patient samples, calculate the median of the category as the methylation level of the category, and use 'Wilcox.test' to calculate respectively The difference in methylation between healthy people and breast cancer patients in the training set and test set is statistically significant. If the P value is ⁇ 0.05, it is considered that the methylation marker has significant methylation differences between healthy people and breast cancer patient samples.
  • Seq ID NO: 152 We take Seq ID NO: 152 as an example to show in detail the methylation levels of this methylation marker in breast cancer and healthy people in the training set and test set, as shown in Table 4C below.
  • the methylation marker in the training set and test set There are extremely significant methylation differences between breast cancer and healthy people.
  • the P value of the training set 'Wilcox.test' is 6.0E-7
  • the P value of the test set 'Wilcox.test' is 7.8E-04.
  • This example uses 79 methylation markers to construct a logistic regression machine learning model to identify plasma samples from healthy people and breast cancer patients.
  • TestPred AllModel.predict_proba(TestData)[:,1], where TestData is the test set data, and TestPred is the model prediction score. Use the prediction score to determine whether the sample is correct according to the above threshold. Is breast cancer to be judged.
  • the distribution of model prediction scores in the training set and test set is shown in Figure 4A. It can be seen from the figure that there is a significant difference in the model scores of breast cancer and healthy people samples.
  • the ROC curve is shown in Figure 5A.
  • the AUC of the model for distinguishing breast cancer from healthy people is 0.992
  • the AUC of the test set is 0.935.
  • the threshold is set to 0.362 based on the training set data. If the value is greater than this value, it is breast cancer, and vice versa. Under this threshold, the test set accuracy is 0.825, the specificity is 0.737, and the sensitivity is 0.905.
  • the specific data are shown in Table 5A.
  • This model can better distinguish breast cancer plasma samples from healthy human plasma samples, and can be used for early screening of breast cancer.
  • TestPred AllModel.predict_proba(TestData)[:,1], where TestData is the test set data, and TestPred is the model prediction score. Use the prediction score to determine whether the sample is correct according to the above threshold. Is breast cancer to be judged.
  • the distribution of model prediction scores in the training set and test set is shown in Figure 4B. It can be seen from the figure that there is a significant difference in the model scores of breast cancer and healthy people samples.
  • the ROC curve is shown in Figure 5B.
  • the AUC of the model for distinguishing breast cancer from healthy people is 0.995
  • the AUC of the test set is 0.962.
  • the threshold is set to 0.440 based on the training set data. If the value is greater than this value, it is breast cancer, and vice versa. Under this threshold, the accuracy of the test set is 0.900, the specificity is 0.842, and the sensitivity is 0.952.
  • the specific data are shown in Table 5B.
  • This model can better distinguish breast cancer plasma samples from healthy human plasma samples, and can be used for early screening of breast cancer.
  • TestPred AllModel.predict_proba(TestData)[:,1], where TestData is the test set data, and TestPred is the model prediction score. Use the prediction score to determine whether the sample is correct according to the above threshold. Is breast cancer to be judged.
  • the distribution of model prediction scores in the training set and test set is shown in Figure 4C. It can be seen from the figure that there is a significant difference in the model scores of breast cancer and healthy people samples.
  • the ROC curve is shown in Figure 5C.
  • the AUC of the model for distinguishing breast cancer from healthy people is 0.975
  • the AUC of the test set is 0.932.
  • the threshold is set to 0.505 based on the training set data. If the value is greater than this value, it is breast cancer, and vice versa. At this threshold, the test set accuracy is 0.875, specificity is 0.789, and sensitivity is 0.952, see Table 5C.
  • This model can effectively distinguish breast cancer plasma samples from healthy human plasma samples, and can be used for early screening of breast cancer.
  • this example selected Seq ID NO: 1, Seq ID NO: 4, Seq ID NO: 9, and Seq ID NO: 10 from all 22 methylation markers. , Seq ID NO: 15, Seq ID NO: 17, a total of 6 methylation markers to construct a new machine learning model Sub1.
  • the method of constructing the machine learning model is the same as in Example 2, but only 6 methylation markers in the random methylation marker combination 1 are used.
  • This model is in the training set and test set.
  • the model score is shown in Figure 6A
  • the ROC curve of the model is shown in Figure 7A. It can be seen that in the training set and test set of this model, the scores of breast cancer samples are significantly different from the scores of healthy people.
  • the AUC of the training set of this model is 0.944
  • the AUC of the test set is 0.912.
  • the threshold is set to 0.609
  • the test set is accurate
  • the accuracy was 0.750
  • the specificity was 0.895
  • the sensitivity was 0.619.
  • the specific data are shown in Table 5A, which illustrates the good performance of this combined model.
  • this example selected Seq ID NO: 67, Seq ID NO: 68, Seq ID NO: 75, and Seq ID NO: 80 from all 22 methylation markers. , Seq ID NO: 84, Seq ID NO: 88, a total of 6 methylation markers to construct a new machine learning model Sub1.
  • the method of constructing the machine learning model is the same as in Example 2, but only 6 methylation markers in the random methylation marker combination 1 are used.
  • the model scores of the model in the training set and the test set are shown in Figure 6B.
  • the ROC of the model The curve is shown in Figure 7B. It can be seen that in the training set and test set of this model, the scores of breast cancer samples are significantly different from the scores of healthy people.
  • the AUC of the training set of this model is 0.930, and the AUC of the test set is 0.867.
  • the threshold is set to 0.541
  • the test set is accurate
  • the accuracy was 0.775
  • the specificity was 0.842
  • the sensitivity was 0.714.
  • the specific data are shown in Table 5B, which illustrates the good performance of this combined model.
  • this example selected Seq ID NO: 142, Seq ID NO: 149, Seq ID NO: 153, and Seq ID NO: 155 from all 35 methylation markers. , Seq ID NO: 162, Seq ID NO: 164, Seq ID NO: 165, a total of 7 methylation markers were used to construct a new machine learning diagnostic model Sub1.
  • the method of constructing the machine learning model is the same as in Example 2, but only 7 methylation markers in random methylation marker combination 1 are used.
  • the model scores of this model in the training set and test set are shown in Figure 6C.
  • the ROC of this model The curve is shown in Figure 7C. It can be seen that in the training set and test set of this model, the scores of breast cancer samples are significantly different from the scores of healthy people.
  • the AUC of the training set of this model is 0.884, and the AUC of the test set is 0.847. When the threshold is set to 0.445 When , the accuracy of the test set was 0.775, the specificity was 0.737, and the sensitivity was 0.810, as shown in Table 5C, which illustrates the good performance of the model.
  • This example uses another random methylation marker combination: Seq ID NO: 2, Seq ID NO: 3, Seq ID NO: 6, Seq ID NO: 12, Seq ID NO: 14, Seq ID NO: 16 , Seq ID NO: 20, a total of 7 methylation markers were used to construct the machine learning model Sub2.
  • the model construction method is also consistent with Example 2.
  • the model scores of the model in the training set and test set are shown in Figure 8A, and the ROC curve is shown in Figure 9A. It can be seen from the figure that in the training set and test set of this model, the score of breast cancer samples is significantly higher than that of healthy people.
  • the AUC of the training set of this model is 0.935, and the AUC of the test set is 0.852.
  • the threshold is set to 0.604
  • the accuracy of the test set is 0.700
  • the specificity is 0.789
  • the sensitivity is 0.619.
  • the specific data is shown in Table 5A, which can also be better distinguished. Breast cancer and normal people.
  • This example uses another random methylation marker combination: Seq ID NO: 69, Seq ID NO: 76, Seq ID NO: 78, Seq ID NO: 79, Seq ID NO: 82, Seq ID NO: 83 , Seq ID NO: 85, a total of 7 methylation markers were used to construct the machine learning model Sub2.
  • the model construction method is also consistent with Example 2.
  • the model is tested on the training set and test
  • the lumped model scores are shown in Figure 8B and the ROC curves are shown in Figure 9B. It can be seen from the figure that in the training set and test set of this model, the score of breast cancer samples is significantly higher than that of healthy people.
  • the AUC of the training set of this model is 0.910, and the AUC of the test set is 0.875.
  • the threshold is set to 0.513
  • the accuracy of the test set is 0.850
  • the specificity is 0.789
  • the sensitivity is 0.905.
  • the specific data is shown in Table 5B, which can also be better distinguished. Breast cancer and normal people.
  • This example uses another random methylation marker combination: Seq ID NO: 136, Seq ID NO: 137, Seq ID NO: 146, Seq ID NO: 147, Seq ID NO: 152, Seq ID NO: 160 , a total of 6 methylation markers were used to construct the machine learning model Sub2.
  • the model construction method is also consistent with Example 2.
  • the model scores of the model in the training set and test set are shown in Figure 8C, and the ROC curve is shown in Figure 9C. It can be seen from the figure that in the training set and test set of this model, the score of breast cancer samples is significantly higher than that of healthy people.
  • the AUC of the training set of this model is 0.849, and the AUC of the test set is 0.865.
  • the threshold is set to 0.447, the accuracy of the test set is 0.800, the specificity is 0.632, and the sensitivity is 0.952. See Table 5C. It can also distinguish breast cancer well. and normal people.
  • the inventor of the present application found that the methylation level of a single methylation marker among the 22 methylation markers also has a good classification effect.
  • the effect of a single methylation marker in distinguishing breast cancer from healthy people is shown in Table 6A.
  • Seq ID NO: 14 if this methylation marker is used alone to build a machine learning model, the model training set AUC is 0.880 and the test set AUC is 0.962.
  • the threshold is set to 0.532, the test set accuracy is 0.875.
  • the specificity was 0.789, the sensitivity was 0.952, and the classification effect was obvious.
  • the inventor of the present application found that the methylation level of a single methylation marker among the 22 methylation markers also has a good classification effect.
  • the effect of a single methylation marker in distinguishing breast cancer from healthy people is shown in Table 6B.
  • Seq ID NO: 75 if this methylation mark is used alone
  • the AUC of the model training set is 0.882
  • the AUC of the test set is 0.774.
  • the threshold is set to 0.534
  • the accuracy of the test set is 0.725
  • the specificity is 0.684
  • the sensitivity is 0.762
  • the classification effect is obvious.
  • the inventor of the present application found that among the 35 methylation markers, the methylation level of a single methylation marker also has a good classification effect.
  • the effect of a single methylation marker in distinguishing breast cancer from healthy people is shown in Table 6C.
  • Seq ID NO: 152 if this methylation marker is used alone to build a machine learning model, the model training set AUC is 0.792 and the test set AUC is 0.802.
  • the threshold is set to 0.533, the test set accuracy is 0.800.
  • the specificity was 0.684, the sensitivity was 0.905, and the classification effect was obvious.
  • This application screened out 79 methylation markers for breast cancer.
  • the machine learning diagnostic model constructed based on the methylation levels of these methylation markers can better distinguish breast cancer from healthy people and provide early screening for breast cancer. Check is of great significance.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne l'utilisation d'un marqueur de méthylation dans le diagnostic du cancer du sein ou la prédiction de risques de cancer du sein. L'invention concerne également l'utilisation d'un réactif pour détecter le niveau de méthylation d'un marqueur dans la préparation d'un kit ou d'un microréseau pour diagnostiquer un cancer du sein ou prédire des risques de cancer du sein chez des individus. L'invention concerne en outre un kit ou un microréseau pour diagnostiquer un cancer du sein ou prédire des risques de cancer du sein chez des individus.
PCT/CN2023/111009 2022-08-04 2023-08-03 Utilisation d'un marqueur dans le diagnostic du cancer du sein ou la prédiction de risques de cancer du sein Ceased WO2024027796A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN202210931191.8A CN117587121A (zh) 2022-08-04 2022-08-04 标志物在诊断乳腺癌或预测乳腺癌风险中的用途
CN202210931401.3A CN117660634A (zh) 2022-08-04 2022-08-04 标志物在诊断乳腺癌或预测乳腺癌风险中的用途
CN202210931193.7 2022-08-04
CN202210931193.7A CN117660633A (zh) 2022-08-04 2022-08-04 标志物在诊断乳腺癌或预测乳腺癌风险中的用途
CN202210931191.8 2022-08-04
CN202210931401.3 2022-08-04

Publications (1)

Publication Number Publication Date
WO2024027796A1 true WO2024027796A1 (fr) 2024-02-08

Family

ID=89848544

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/111009 Ceased WO2024027796A1 (fr) 2022-08-04 2023-08-03 Utilisation d'un marqueur dans le diagnostic du cancer du sein ou la prédiction de risques de cancer du sein

Country Status (2)

Country Link
TW (1) TW202413655A (fr)
WO (1) WO2024027796A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101298629A (zh) * 2008-06-20 2008-11-05 中南大学 Lrrc4基因启动子区甲基化检测在脑胶质瘤诊断中的应用及其检测系统
US20120172238A1 (en) * 2009-09-22 2012-07-05 Cold Spring Harbor Laboratories Method and compositions for assisting in diagnosing and/or monitoring breast cancer progression
CN107034295A (zh) * 2017-06-05 2017-08-11 天津医科大学肿瘤医院 用于癌症早期诊断和危险度评价的dna甲基化指标及其应用
US20170283886A1 (en) * 2014-09-15 2017-10-05 Garvan Institute Of Medical Research Methods for Diagnosis, Prognosis and Monitoring of Breast Cancer and Reagents Therefor
US20190256921A1 (en) * 2016-05-04 2019-08-22 Queen's University At Kingston Cell-free detection of methylated tumour dna

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101298629A (zh) * 2008-06-20 2008-11-05 中南大学 Lrrc4基因启动子区甲基化检测在脑胶质瘤诊断中的应用及其检测系统
US20120172238A1 (en) * 2009-09-22 2012-07-05 Cold Spring Harbor Laboratories Method and compositions for assisting in diagnosing and/or monitoring breast cancer progression
US20170283886A1 (en) * 2014-09-15 2017-10-05 Garvan Institute Of Medical Research Methods for Diagnosis, Prognosis and Monitoring of Breast Cancer and Reagents Therefor
US20190256921A1 (en) * 2016-05-04 2019-08-22 Queen's University At Kingston Cell-free detection of methylated tumour dna
CN107034295A (zh) * 2017-06-05 2017-08-11 天津医科大学肿瘤医院 用于癌症早期诊断和危险度评价的dna甲基化指标及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN, LING ET AL.: "Down-regulation of tumor suppressor gene FEZ1/LZTS1 in breast carcinoma involves promoter methylation and associates with metastasis", BREAST CANCER RES TREAT., vol. 116, no. 3, 31 August 2009 (2009-08-31), pages 471 - 478, XP019727895 *
WANG, XINXIN ET AL.: "Loss of Leucine Zipper Putative Tumor Suppressor 1 (LZTS1) Expression Contributes to Lymph Node Metastasis of Breast Invasive Micropapillary Carcinoma", PATHOL. ONCOL. RES., vol. 21, 27 March 2015 (2015-03-27), pages 1021 - 1026, XP035530316, DOI: 10.1007/s12253-015-9923-x *

Also Published As

Publication number Publication date
TW202413655A (zh) 2024-04-01

Similar Documents

Publication Publication Date Title
CN113557308B (zh) 检测子宫内膜癌
EP3314016B1 (fr) Procédés de diagnostic du cancer de la vessie
CN113811622A (zh) 在血浆中检测胰腺导管腺癌
CN114651073A (zh) 结肠直肠癌和/或进行性腺瘤的检测
CN108866191B (zh) 基于甲基化修饰的肿瘤标记物stamp-ep2
EP3904515A1 (fr) Marqueur tumoral stamp-ep3 basé sur une modification de méthylation
KR20150067151A (ko) 암을 스크리닝하는 방법
CN113308544B (zh) 用于dna甲基化检测的试剂及食管癌检测试剂盒
JP6395131B2 (ja) 肺癌に関する情報の取得方法、ならびに肺癌に関する情報を取得するためのマーカーおよびキット
WO2024056008A1 (fr) Marqueur de méthylation pour identifier un cancer et son utilisation
EP3983566A1 (fr) Méthodes de détection et de prédiction du cancer du sein
CN115572765A (zh) 一组肿瘤检测标志物及其用途
CN120457220A (zh) 使用dna甲基化标志物对晚期腺瘤和/或结直肠癌进行分层和早期检测的方法
CN116555422A (zh) 肺癌甲基化标志物、检测试剂盒及其应用
EP3625370A1 (fr) 1 biomarqueurs épigénétiques composites pour criblage, diagnostic et pronostic précis du cancer colorectal
CN116555423A (zh) 肺癌甲基化标志物组合、检测产品及其应用
US11542559B2 (en) Methylation-based biomarkers in breast cancer screening, diagnosis, or prognosis
JP2025016597A (ja) Ｄｎａメチル化バイオマーカーの組み合わせ、検出方法および試薬キット
CN117660633A (zh) 标志物在诊断乳腺癌或预测乳腺癌风险中的用途
CN117660634A (zh) 标志物在诊断乳腺癌或预测乳腺癌风险中的用途
WO2024027796A1 (fr) Utilisation d'un marqueur dans le diagnostic du cancer du sein ou la prédiction de risques de cancer du sein
EP2450455B1 (fr) Procédé permettant de déterminer si des cellules épithéliales d'origine cancéreuses sont présentes ou pas dans un échantillon biologique et nécessaire associés
CN115678990A (zh) 标志物在预测结直肠癌的复发和/或转移风险中的用途
CN115772565A (zh) 用于辅助检测肺癌体细胞egfr基因突变的甲基化位点及其应用
WO2021255460A1 (fr) Méthodes de détection et de prédiction de néoplasie épithéliale cervicale de stade 3 (cin3) et/ou de cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23849500

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 23849500

Country of ref document: EP

Kind code of ref document: A1