EP1337668A2 - Procede de detection d'etats de methylation afin de permettre le diagnostic toxicologique - Google Patents

Procede de detection d'etats de methylation afin de permettre le diagnostic toxicologique

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Publication number
EP1337668A2
EP1337668A2 EP01996625A EP01996625A EP1337668A2 EP 1337668 A2 EP1337668 A2 EP 1337668A2 EP 01996625 A EP01996625 A EP 01996625A EP 01996625 A EP01996625 A EP 01996625A EP 1337668 A2 EP1337668 A2 EP 1337668A2
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EP
European Patent Office
Prior art keywords
protein
precursor
kinase
subunit
factor
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.)
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EP01996625A
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German (de)
English (en)
Inventor
Alexander Olek
Christian Piepenbrock
Kurt Berlin
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Epigenomics AG
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Epigenomics AG
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Publication of EP1337668A2 publication Critical patent/EP1337668A2/fr
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/142—Toxicological screening, e.g. expression profiles which identify toxicity
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the methylation states of toxicologically relevant genes are determined and the data acquired in this process are summarized for methylation patterns.
  • comprehensive prognostic statements can be made about the toxicological properties of substances.
  • a method is to be presented which enables the methylation positions of the genes to be examined to be analyzed to a large extent.
  • P- er ⁇ P- 1 P- er c P ⁇ ⁇ sQ 02 O C ⁇ ⁇ Q ⁇ o; r et P 1 CQ es s rt C ⁇ es rt iQ Q-DJ rt $. ⁇ rt P rt N es C ⁇ ⁇ PP "CQ rt es P 1 u • ⁇ > Q ⁇ N er N ⁇ md I- 1 o P- CU ⁇ g ⁇ » c CL vQ P- ⁇ P- P- P- ⁇ ! P 1 P CL ⁇ er n ⁇ • ⁇ P ⁇ d
  • P- t ⁇ CQ s P- ⁇ ⁇ es es P- ⁇ ⁇ P- ⁇ • ö 1 rt ⁇ ⁇ P es iQ ⁇ 1 co ⁇ ⁇ ⁇ C ⁇ ⁇ 3 er ⁇ P- 1 P 1 • rt ⁇ 1 es C ⁇ ⁇ P ⁇ es P- 3 er ⁇ co P- "d 1 1 es 1 rt P, rt f Hi
  • Fluorescence-labeled probes have been used in many cases for scanning an immobilized DNA array.
  • the simple attachment of Cy3 and Cy5 dyes to the 5-OH of the respective probe is particularly suitable for fluorescent labels.
  • the fluorescence of the hybridized probes is detected, for example, using a confocal microscope.
  • the dyes Cy3 and Cy5, among many others, are commercially available.
  • Matrix-assisted laser desorption / ionization mass spectrometry is a very powerful development for the analysis of biomolecules (Kara, M. and Hillenkamp, F. (1988), Laser desorption ionization of proteins with molecular asses exeeding 10,000 daltons. Anal. Che. 60: 2299-2301).
  • An analyte is in embedded a light absorbing matrix. The matrix is evaporated by a short laser pulse and the analyte molecule is thus transported unfragmented into the gas phase. The ionization of the analyte is achieved by collisions with matrix molecules.
  • An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, ions are accelerated to different extents. Smaller ions reach the detector earlier than larger ones.
  • Genomic DNA is obtained by standard methods from DNA from cell, tissue or other test samples. This standard methodology can be found in references such as Fritsch and Maniatis eds. , Molecular Cloning: A Laboratory Manual, 1989.
  • the present invention is intended to provide a method which is suitable for the diagnosis of methylation states of genes with toxicological relevance.
  • the invention is based on the finding that cytosine methylation states in particular are particularly suitable for diagnosing changes in the expression of genes with toxicological relevance.
  • the present invention describes a method for
  • a genomic DNA sample is preferably chemically treated in such a way that at the 5'-position unmethylated cytosine bases are converted into uracil, thymine or another base which is not similar to cytosine in terms of hybridization behavior. This is understood below as chemical pretreatment.
  • the base sequence of a part of the chemically treated DNA is determined and the methylation states characteristic of the sample are inferred.
  • fragments are preferably first amplified from the chemically pretreated genomic DNA using preroligonucleotides.
  • More than 10 different fragments that are 100-2000 base pairs long are preferably amplified.
  • the amplification is carried out by means of the polymerase chain reaction (PCR), a thermostable DNA polymerase preferably being used.
  • PCR polymerase chain reaction
  • the set of primer oligonucleotides comprises at least two oligonucleotides, the sequences of which are each reversely complementary or identical to a section of the sequences of the genes to be examined that is at least 18 base pairs long.
  • the preroligonucleotides are preferably characterized in that they contain no CpG dinucleotide.
  • At least one of the two primer oligonucleotides used to amplify a specific section of the chemically pretreated DNA preferably contains identifiable markings.
  • the labels of the amplified products are fluorescent labels.
  • the labels of the amplificates are radionuclides.
  • the markings of the amplificates are detachable molecular fragments with a typical mass, which are detected in a mass spectrometer.
  • the amplicons, fragments of the amplicons or probes complementary to the amplicons are detected in the mass spectrometer.
  • the fragments generated have a single positive or negative net charge for better detectability in the mass spectrometer.
  • MALDI matrix-assisted laser desorption / ionization Mass spectrometry
  • ESI electrospray mass spectrometry
  • At least one primer oligonucleotide is bound to a solid phase during the amplification.
  • the probe oligonucleotides or PNA oligomers are preferably bound to a solid phase at defined locations.
  • oligonucleotides and / or PNA oligomer sequences are arranged on a flat solid phase in the form of a rectangular or hexagonal grid.
  • the solid phase surface preferably consists of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold.
  • the amplificates are hybridized to a set of at least 10 oligonucleotide or the PNA oligomer probes.
  • the amplificates serve as samples that hybridize to oligonucleotides previously bound to a solid phase.
  • Hybridization in the sense of this invention is to be understood as binding with the formation of a duplex structure of an oligonucleotide to a completely complementary sequence in the sense of the Watson-Crick base pairings in the sample DNA. The non-hybridized fragments are then removed.
  • Said oligonucleotides comprise at least one base sequence with a length of 13 nucleotides, which is reverse complementary or identical to a section of the base sequences of the genes to be examined.
  • the cytosine of the CpG dinucleotide is the 5th to 9th nucleotide from the 5 'end viewed from the 13s.
  • the said PNA oligomers comprise at least one base sequence with a length of 9 nucleotides, which is reverse complementary or identical to a section of the base sequences of the genes to be examined, which contains at least one CpG dinucleotide.
  • the cytosine of the CpG dinucleotide is the 4th to 6th nucleotide as seen from the 5 'end of the 9 mer.
  • the non-hybridized amplificates are removed.
  • the hybridized amplificates are detected.
  • markings attached to the amplifiers can be identified at any position of the solid phase at which an oligonucleotide sequence is located.
  • the method is preferably used to diagnose and / or predict adverse events for patients or individuals, these adverse events being associated with the diagnosis of toxicologically significant parameters.
  • the method is used for the diagnosis and / or prognosis of adverse events for patients or individuals, these adverse events being related to the diagnosis of toxicologically important parameters.
  • the set of genes to be examined includes at least one of the genes in Tab. 1 genes or sequences that are identical in the area of the exons or at least 85% homologous to those in Tab. 1 genes listed.
  • Lactotransferrin precursor Lactoferrin precursor
  • Apolipoprotein A-I precursor (APOAI) X00566
  • Apolipoprotein A-II precursor (APOAI I) X00955 Apolipoprotein C-III precursor (APOCIII) X01388
  • CSF1 CSF
  • CSF CSF
  • FAC1 familial intrahepatic cholestasis 1 protein
  • VEGFD C-FOS-induced growth factor
  • Complement component 4 binding protein alpha C4B binding protein; C4BPA
  • IGF2 Insulin-like growth factor II
  • GM-CSF CSF2 M11220 epidermal growth factor precursor (EGF);
  • GGFHPP2 Glial growth factor 2 precursor
  • T cell specific Rantes protein precursor sis delta
  • small inducible cytokine A5 SCYA5
  • SYA5 small inducible cytokine A5
  • IGFBP1 Placenta protein 1 (PP12) M31145
  • VEGF Vascular Endothelial Growth Factor Precursor
  • VPF Vascular Permeability Factor
  • HGF Hepatocyte growth factor
  • Thymosin beta-10 TMSB10; THYB10; PTMB10 M92381
  • Interferon gamma-induced protein precursor (gamma-IPIO) X02530
  • OX40 ligand OX40L
  • GP34 tax-transcriptionally activated glycoprotein 1
  • TGF-beta3 transforming growth factor beta 3 J03241
  • Delta-like protein precursor U15979; Z12172 Insulin-like growth factor IA precursor (IGF1A); IGFBP1; So atomedin C + insulin-like growth factor I (IGF1) 27544 + M37484 CG chemokine-eotaxin precursor; eosinophil chemotactic protein; small inducible cytokine All (SCYA11) D49372; Z75669;
  • Interleukin-1 receptor antagonist protein precursor IL-1RA; IRAP
  • MIC1 macrophage inhibitor cytokine 1
  • Eosinophil Gramule Major Basic Protein Precursor (MBP); Affiliation-associated major-basic protein; bone marrow proteoglycan 2 Y00809 Insulin-like growth factor-binding protein 3 precursor (IGF-binding protein 3;
  • IGFBP3 IGFBP3
  • IBP3 IBP3
  • M31159 M35878 cellular retinoic acid binding protein II (CRABP2)
  • CRABP2 cellular retinoic acid binding protein II
  • Corticotropin-releasing factor precursor Corticotropin release factor (CRF); Corticotropin release hormone (CRH) V00571
  • Interferon gamma precursor IFN-gamma; IFNG; Immune interferon X01992; M29383
  • Interleukin-2 precursor IL-2
  • T cell growth factor TCGF
  • Interleukin-1 alpha precursor IL-1 alpha
  • ILlA Interleukin-1 alpha precursor
  • IL-4 Hematopoietin-1 X02851 interleukin-4 precursor
  • BSF-1 B cell stimulation factor 1
  • Interleukin-6 precursor IL-6
  • BCF2 B cell stimulation factor 2
  • IFNB2 Interferon beta-2
  • Hybridoma growth factor X04602 14584 Interleukin-5 precursor (IL-5); T cell
  • TRF substitution factor
  • IL-12B cytotoxic lymphocyte maturation
  • NKSF2 M65290 Interleukin-12 alpha subunit precursor
  • IL-12A cytotoxic lymphocyte maturation
  • NKSF1 M65291 Pancreatitis Associated Protein 1 Precursor D13510
  • Alpha-1-acid-glycoprotein-1 precursor (AGP1);
  • Prostaglandin G / H synthasel (PGH synthase-1;
  • 5-hydroxytryptamine ID receptor (5-HT-ID; HTR1D); Serotonin receptor M89955
  • Dopamine beta hydroxylase (DBH); Dopamine beta monooxygenase precursor X13255
  • EPOR Erythropoietin Receptor M60459 cation-independent mannose-6-phosphate receptor precursor (Cl Man-6-P receptor; CI-MPR); Insulin- like growth factor II receptor (IGFR II) Y00285; J03528
  • ACTRIIA Activin receptor type II precursor
  • Retinoid X receptor GAMMA RXR-GAMMA
  • TEZ transcriptional enhancer factor
  • LDL receptor Low-density lipoprotein receptor
  • Sulfonylurea receptor 2A (SUR2A) AF061323
  • SUR Sulfonylurea receptor
  • CTNNA1 Alphal catenin
  • Cadherin-associated Cadherin Alphal catenin (CTNNA1); Cadherin-associated
  • Integrin-alpha-9 (ITGA9); Integrin alpha RLC D25303; L24158 interzullar adhesion molecule 1 precursor (ICAM1); Main group rhinovirus receptor; CD54 antigen J03132
  • E-selectin precursor SELE
  • Endothelial Leukocyte Adhesion Molecule 1 ELAMl
  • Leukocyte endothelial cell adhesion molecule 2 LCAM2
  • CD62E antigen M30640 NADH-ubiquinone dehydrogenase-1-beta subcomplex 7 18kDa subunit (NDUFB7)
  • Complex-I-B18 CI-B18
  • Cell Adhesion Protein SQM1 M33374 Neural Cadherin Precursor N-Cadherin; NCAD
  • Cadherin 2 CDH2 M34064; X57548; X54315; S42303
  • Integrin alpha 5 (ITGA5); VLA5; CD49E antigen X06256
  • Fibronectin receptor beta subunit FNRB
  • Integrin alpha L (ITGAL); Leukozytahpsions-
  • LFA1 CDllA antigen Y00796 cadherin-6 precursor (CDH6); Kidney-cadherin
  • Cadherin 12 (CDH12); Brain cadherin precursor (Br-cadherin); neural cadherin 2 (N-cadherin 2) L34057; L33477
  • T-cadherin Cardiac cadherin (H-cadherin) L34058; U59289;
  • Cadherin 3 (CDH3); Placenta-cadherin precursor (P-cadherin; CDHP) X63629
  • CBP-I Placenta anticoagulant protein I
  • PAP-I Placenta anticoagulant protein I
  • PP4 Thromboplastin inhibitor
  • Anticoagulant alpha (VAC-alpha; anchorin CII X12454
  • Aminin alpha 1 subunit precursor (LAMA1);
  • FABP1 Liver Fatty Acid Binding Protein 1
  • LFABP LFABP
  • TNF-alpha stimulated ABC protein TSAP AF027302
  • Organic Cation Transporter Protein 2 (ORCTL2) AF037064 Organic Cation Transporter N2 (OCTN2) AF057164 MRP / Organic Cation Transporter (MOAT-B) AF071202 Adrenoleukodystrophy-Related Protein (ALDR) AJ000327 Skeletal Muscle Adenine Nucleotide (transliterate nucleotide) ANT1); Heart / skeletal muscle ADP / ATP carrier protein isoform Tl; ADP / ATP translocase 1 J02966
  • DDA Down-regulated protein in adenoma
  • UCP3 mitochondrial decoupling protein-3
  • CPTase mitochondrial carnitine palmitoyl transferase-II precursor
  • CPT2 mitochondrial carnitine palmitoyl transferase-II precursor
  • PTT Prostaglandin transporter
  • SLC21A2 dissolved carrier family 21 member 2
  • U70867 mitochondrial decoupling protein 2 U82819
  • Bile salt export pump (BSEP) AF091582
  • Anthracycline resistance associated protein (ERA) X95715 kidney transporter for organic cation X98333
  • MRP3 Multi-resistance associated protein 3
  • Antigen-peptide transporter 2 APT2
  • Peptide delivery factor 2 PSF2
  • TAP2 peptide transporter
  • MLR / TAP ATP-binding cassette subfamily B
  • ABCC3 HLA class II histocompatibility antigen DO-beta
  • Nucleotide sensitive chloride channel 1A Chloride ion current inducer protein (CLCI); Reticulocyte PICLN X91788 Transporter A for neutral amino acid (SATT); Alanine / Serine / Cysteine / Threonine Transporter (ASCT1) L14595 Monocarboxylate Transporter-1 (MCT1) L31801
  • CLCI Chloride ion current inducer protein
  • SATT Reticulocyte PICLN X91788 Transporter A for neutral amino acid
  • ASCT1 Alanine / Serine / Cysteine / Threonine Transporter
  • MCT1 Monocarboxylate Transporter-1
  • Ileal sodium-dependent bile salt transporter ISBT
  • Ileal sodium / taurocholate co-transporting polypeptide NTCP2
  • SLC10A2 U10417 sodium-dependent bile salt cotransporter hepatic natriu / taurocholate co-transporting polypeptide (NTCP)
  • GLYT-1 S70609 Cystic Fibrosis Transmembrane Conductivity Regulator
  • CFTR cAMP-dependent chloride channel M28668 channel-shaped multispecific transporter for organic anion
  • MRP2 Multi-resistance associated protein 2
  • U63970 multi-resistance channel-associated protein
  • Gap junction beta-l protein (connexin 32)
  • Cadherin 1 (CDH1); epithelial cadherin precursor (E-cadherin; CDHE); Uvomorulin (UVO); CAM 120/80 Z13009 smoothed; GX U84401
  • ⁇ phrin type A receptor 2 precursor epithelial cell kinase (ECK); Tyrosine protein kinase receptor ECK M59371 M36395 NADPH cytochrome p450 reductase S90469
  • NCK melanoma cytoplasmic src homolog HSNCK
  • Dual-specificity mitogen-activated protein kinase kinase-1 MAP kinase kinase 1; MAPKK 1; MKK1
  • extracellular signal-regulated kinase 1 ⁇ RK
  • JNK1 c-jun N-terminal kinase 1
  • Mitogen-activated protein kinase p38 MAP kinase p38
  • Cytokine suppressive "anti-inflammatory" drug-binding protein CSAID-binding protein; CSBP
  • MAX MAX interacting protein 2 (MXI2) L35253; L35263
  • Protein kinase C beta I (PKC-beta-1) M27545; X06318
  • Mitogen-activated protein kinase 9 MAP kinase 9; MAPK9; PRKM9; "c-jun" N-terminal kinase 2 (JNK2); JNK55 L31951
  • MAPK / ⁇ RK kinase 6 MAPK / ⁇ RK kinase 6; SAPKK3 U39657 p21 activated kinase gamma (PAK gamma; PAK2);
  • Mitogen-activated protein kinase P38 beta MAP Kinase P38 beta
  • MAPK / ERK kinase kinase 3 (MEK kinase 3; MEKK3) U78876 dual-specificity mitogen-activated protein kinase
  • MAP Kinase Kinase 2 (MAP Kinase Kinase 2; MAPKK 2); ERK activator kinase 2; MAPK / ERK Kinase 2 (MEK2) L11285 dual-specificity mitogen-activated protein kinase
  • MAP Kinase Kinase 5 (MAP Kinase Kinase 5; MAPKK 5) U25265 ribosomal protein S6 kinase II alpha 1 (S6KII-alpha 1); ribosomal S6 kinase 1 (RSK1) L07597 B lymphocyte germ center kinase (GC kinase) U07349
  • Protein tyrosine phosphatase MEG2 (PTPASE-MEG2) M83738 Protein tyrosine phosphatase alpha precursor (R-PTP-alpha; PTPRA; PTPA) M34668 diabetes-associated RAS (RADI) L24564
  • calbindin Avian-type vitamin .D-dependent calcium binding protein (CABP); D-28K X06661
  • Cell marker protein 1 HME1 AF029082 FKBP rapamycin associated protein (FRAP);
  • Zinc finger protein 37 (ZFP37); KRAB-zinc Region
  • CCAAT / enhancer-binding protein epsilon (C / EBP Epsilon; CEBPE) U48866; U48865
  • RPL6 60S ribosomal protein L6
  • TAXREB107 TAX-responsive enhancer element binding protein 107
  • Zinc finger protein 40 (ZNF40); human immunodeficiency virus type I enhancer binding
  • MBP-1 Complex binding protein 1 (MBP-1); positive regulator region II binding factor 1
  • N-oct3 Transcription factor N-oct3; N-oct5A &N-oct5B;
  • hypoxia-inducible factor 1 alpha ARNT interacting protein
  • DNA binding protein inhibitor Id-2 M97796 activating transcription factor 4 (ATF4);
  • Tax-responsive enhancer element B67 (TAXREB67); cAMP response element binding protein 2 (CREB2) D90209
  • HSF1 Heat shock factor protein 1
  • HSTF1 Heat shock transcription factor 1
  • TCF5 TCF5 M64673
  • FK506 binding protein 13 precursor FKBP13
  • FKBP2 Peptidyl prolyl cis trans isomerase (PPIase) M65128 cAMP response element binding protein (CRE-BPl); Transcription factor ATF2; HB16 M31630 cAMP Response Element Binding Protein (CREB) M34356 Initial Growth Response Protein 1 (EGR1); Transcription factor ETR103; KROX24; Zinc finger protein 225 (ZNF225); AT225 X52541; M62829 tristetraproline (TTP); TISll; ZFP36; Growth factor-inducible core protein 475 (NUP475) M92843 purine-rich single-stranded DNA-binding protein alpha (PURA) M96684 transcription factor-relB; I-rel M83221 cyclic AMP-dependent transcription factor ATF-3 (activating factor FACTOR 3) L19871 octamer-binding transcription factor 1 (oct-1; OTF1); Octamer binding protein NF-AI; P
  • PRB binding protein E2F1 Retinoblastoma binding protein 3 (RBBP3); Retinoblasto Associated Protein 1 (RBAP1); PBR3 M96577
  • Retinoic acid receptor alpha Retinoid X receptor alpha (RXRA) X52773
  • Methyl CpG binding protein 2 (MECP2) L37298
  • Precursor (ERP60); 58-kDa microsomal protein;
  • HSC70 interacting protein Progesterone receptor-associated P48 protein U28918
  • PAF-2 Peroxisome assembly factor-2
  • MMP-Xl Membrane type matrix metalloproteinase 1
  • TRIP1 Erythroid potentiating activity (EPA); Fibroblast collagenase inhibitor X03124 alpha-1-antichymotrypsin precursor (ACT) K01500 alpha-1-antitrypsin precursor; alpha-1-protease
  • DBPA DNA binding protein A
  • CSDA Cold Shock Region Protein A
  • Decoy receptor 3 (DCR3) AF104419
  • Replication factor C-36 kDa subunit RRC36
  • Replication factor C-38 kDa subunit RRC38
  • TOP2A DNA topoisomerase II alpha
  • PCNA cyclic nuclear antigen
  • TTT terminal deoxynucleotidyl transferase
  • DNTT M11722 K01919
  • XPG X-ray repair-complementing defective repair in Chinese hamster cells 5" (XRCC5) L20046; X69978
  • TAA Thyroid lupus autoantigen
  • CTCBF CTC box binding factor 85kDa subunit
  • Xeroderma pigmentosum group B complementary Protein XPB
  • ERCC3 "excision repair cross-complementing rodent repair deficiency complementation group 3"(ERCC3)
  • basilar transcription factor 2-89kDa subunit BTF2-p89; TFIIH-89kDa subunit
  • M31899 Ku-70kDa subunit ATP-dependent DNA helicase II 70kDa subunit
  • Lupus-ku autoantigen protein P70 Thyroid lupus auto antigen
  • CTC Box Binding Factor 75kDa Subunit CTC75 M32865; S38729
  • Ubiquitin-conjugating enzyme E2 17-kDa UBE2A Ubiquitin-protein ligase; Ubiquitin carrier protein; HR6A M74524
  • POLA DNA polymerase alpha catalytic subunit
  • MGMT 6-O-methylguanine DNA methyl transferase
  • XPD methylated DNA protein cysteine methyl transferase
  • XRCC221 Xeroderma pigmentosum group D complementary protein
  • HHR23B Xeroderma Pigmentosum Group C Repair Complementing Complex 58-kDa ProteinD21090 HHR23A; UV excision repair protein Protein RAD23A D21235 DNA-dependent protein kinase (DNA-PK) +
  • DNA-PK catalytic subunit U35835 + U47077
  • TDG thymine DNA glycosylase
  • Uracil DNA glycosylase precursor (UNG1) X15653
  • DNA (apurine or apyrimidine site) lyase
  • APE1 AP endonuclease 1
  • APEX apurinic / apyrimidine endonuclease
  • APEN APEX nuclease
  • REF1 X59764 X66133
  • DNase I Deoxyribonuclease I M55983 dual specificity protein phosphatase 9;
  • PRAD1 Parathyroid adenomatosis 1
  • CDK7 Protein kinase 7
  • CAK CDK activating kinase
  • CDK2 Cyclin-dependent protein kinase 2
  • Mitogen-activated protein kinase 2 (MAP kinase 2; MAPK 2); p42-MAPK M84489
  • Mitogen-activated protein kinase 3 (MAPK3; PRKM3);
  • MAPK1 extracellular signal-driven
  • ERK1 ERK1
  • Microtubule-associated protein-2 ERK1
  • MAP kinase 3 (MAPK3; p97-MAPK); PRKM5 X80692
  • CDK4 CDK4
  • CDC2 Cell division control protein 2 homolog
  • p34 protein kinase Cyclin-dependent kinase 1 (CDKl) X05360 extracellular signal-driven kinase 4 (ERK4)
  • ERK4 extracellular signal-driven kinase 4
  • MAPK4 MAP kinase 4
  • PRKM4 PRKM4 X59727
  • CDK5 Cell division protein kinase 5
  • tau protein tau protein
  • PSSALRE X66364 extracellular signal-driven kinase 6 (ERK6); Stress-activated protein kinase-3; Mitogen-activated protein kinase p38 gamma; (MAP kinase p38 gamma) X79483
  • CDKN1A Cyclin-dependent kinase inhibitor 1A
  • MDA6 Melanoma differentiation-associated protein 6
  • CDK-interacting protein 1 (CIP1); AF1; SDI1 U09579; L25610 weelHu CDK tyrosine 15 kinase; wee-1-like protein kinase U10564
  • CDKN2B Cyclin-dependent kinase 4 inhibitor 2B
  • pl4-INK4B Polytumor suppressor 2 (MTS2) U17075; L36844
  • DNA binding protein inhibitor ID-1 DNA binding protein inhibitor ID-1; Id-IH D13889
  • Prothymosin alpha (PROT-alpha; PTMA) M26708
  • GEF Growth inhibitor factor
  • MT-III Metallothionein-III
  • M93311 Growth inhibitor factor
  • HSP40 heat shock protein 1
  • DNAJ protein 40kDa heat shock protein 1 (HSP40); DNAJ protein
  • HDJ1 Homolog 1 (HDJ1; DNAJ1) D49547
  • HSP60 heat shock protein
  • HSPDl 60kDa heat shock protein
  • HSP90A heat shock protein A
  • HSP86 HSPCA X07270 27kDa heat shock protein
  • SRP27 Stress responsive protein 27
  • 24kDa estrogen-controlled protein HSPB1 X54079
  • Cytochrome P450 IIA6 (CYP2A6) + CYP2A7 + CYP2A13 + CYP2A7PT + CYP2A7PC M33318; M33316 + U22029 + U22030 + U22044
  • Cytochrome P450 IIB6 (CYP2B6) + CYP2B3 M29874; J02864 Cytochrome P450 IIIA3 (CYP3A3) + CYP3A4 + CYP3A5 + CYP3A7 M13785 + M18907 + J04813 + D00408 Cytochrome P450 IVA11 (CYP4A11) L04751
  • Cytochrome P450 VIIA1 (CYP7A1) X56088
  • DAMOX D-amino acid oxidase
  • Cytochrome P450 HEI CYP2E1 J02625 Cytochrome P450 IIF1 (CYP2F1) J02906
  • Cytochrome P450 IVB1 (EC 1.14.14.1) (P450-HP) J02871 cytochrome P450 IA2 (P450-P3) (P450-4) Z00036 plasma glutathione peroxidase precursor (GPXP; GPX3) D00632; X58295 natural killer cell enhancing factor (NKEFB) + thiol-specific antioxidant protein (TSA); Thioredoxin peroxidase 1 (TDPX1); Thioredoxin-dependent peroxide reductase 1 L19185 + Z22548; X82321
  • TDPX2 Thioredoxin peroxidase 2
  • TDPX2 Thioredoxin-dependent peroxide reductase 2
  • PAG Proliferation-associated gene
  • NKEFA natural killer cell enhancing factor A
  • GRase glutathione reductase
  • GSR GSR
  • GR X15722 microsomal glutathione S-transferase 12
  • Glutathione-S-transferase pi GSTP1; GST3 X08058; M24485 glutathione peroxidase (GSHPX1; GPX1) Y00483; M21304 glutathione-S-transferase theta 1 (GSTTl) X79389 methallothionein IH (MT1H); MetallothineinO (MT0) + MT1I; MT2 + MT1L + MT1R X64177 + X97260 + X76717 + X97261
  • Glutathione peroxidase gastrointestinal GSHPX-GI
  • Glutathione peroxidase-related protein 2 GPRP
  • X53463 heme oxygenase 1 HOl
  • Glutathione-S-transferase ul (GSTM1; GST1); HB
  • Glutathione-S-transferase AI GTH1; GSTA1; HA-
  • Subunit 1 GST-epsilon M25627 Glutathione-S-Transferase (GST) homolog U90313
  • Glutathione synthetase GSH synthetase
  • NAD H-dehydrogenase
  • Quinone reductase Quinone reductase
  • DT diaphorase Azoreductase
  • Phylloquinone reductase Phylloquinone reductase
  • Transcript 1 M60974 tumor necrosis factor alpha precursor (TNF-alpha;
  • Lymphotoxin alpha precursor (LT-alpha); Tumor-alpha
  • Necrosis factor beta (TNF-beta; TNFB) D12614 fas antigen ligand (FASL); Apoptosis antigen ligand (APTL; APT1LG1); TNFSF6 D38122; U08137
  • TNFR Tumor necrosis factor receptor
  • TNFR2 Tumor necrosis
  • TBP2 Factor binding protein 2
  • TFR1 Tumor necrosis factor receptor 1
  • TBP1 Tumor necrosis factor binding protein 1
  • CD120A-TNFR1 Tumor necrosis factor receptor 1
  • TBP1 Tumor necrosis factor binding protein 1
  • Antigen M33294 fasL receptor Apoptosis-supporting surface
  • Retinoic acid receptor beta (RXR-beta; RXRB) M84820; X63522;
  • CD27BP (Siva) U82938 Tumor Necrosis Factor Ty ⁇ -1 Receptor Associated
  • Interleukin-1 beta convertase precursor IL-1BC
  • IL-1 beta converting enzyme ICE
  • p45 IL-1 beta converting enzyme
  • Caspase-6 precursor (CASP6); Cysteine protease MCH2 isoforms alpha + beta U20536 + U20537 Caspase-4 precursor (CASP4); ICH-2 protease; TX protease; ICE (REL) -II + caspase-5 precursor
  • TNF-related apoptosis inducing ligand TRAIL
  • APO-2 ligand APO-2 ligand
  • Caspase-8 precursor (CASP8); ICE-like apoptotic protease 5 (ICE-LAP5); MORTl-associated CED-3 homolog (MACH); FADD homolog ICE / CED-3-like protease (FADD-like ICE; FLICE); apoptotic cysteine protease MCH-5 U60520; U58143;
  • NIP3 U15174 bcl2 homologous antagonist / killer (BAK) Ü23765; U16811;
  • X84213 induced myeloid leukemia cell differentiation protein MCL-1 L08246 BAD protein; bcl-2 binding component 6 (BBC6); bcl-2L8 U66879
  • BAG-1 BCL-2 binding Athanogen-1 (BAG-1); Glucocorticoid receptor-associated protein
  • Interferon-inducible RNA-dependent protein kinase (P68 kinase) M35663; U50648 inducible nitric oxide synthase (INOS); Type II NOS; Hepatocyte NOS (HEP-NOS) L09210
  • CLU Clusterin precursor
  • SP-40 Complement-associated protein SP-40
  • CLI Complement lysis inhibitor
  • APOJ Apolipoprotein J
  • TRPM2 Testosterone Repressed Prostate "Message” 2
  • SGP2 sulfated glycoprotein 2
  • GADD153 Growth style stand & DNA damage inducible Protein 153 (GADD153); DNA damage inducible
  • Inhibitor of apoptosis protein 1 (HIAPl; API1) + IAP homolog C; TNFR2-TRAF Signaling Complex Protein 1;
  • MYC Avian Myelocytomatosis Viral Oncogene Homolog
  • Subunit precursor (PDGFB; PDGF2); Bacaplermin; c-sis X02811 X02744; M12783 M16288 p53 cellular tumor antigen M14694 M14695
  • B-MYB MYB-related protein B
  • MYBL2 Avian Myeloblastosis Viral Oncogene Homologous Type 2 (MYBL2) X13293 Triiodothyronine Receptor; Thyroid hormone receptor
  • THRA1 v-erbA-related protein Protein ear-1 M24898 "jun” proto-oncogene; Avian Sarcoma Virus 17 Oncogene Homolog; Transcription factor AP-1 J04111 Insulin-like growth factor binding protein 2
  • IGFBP2 IGFBP2 M35410 c-myc purine binding transcription factor puf;
  • NDP kinase B Nucleoside diphosphate kinase B (NDP kinase B; NDKB)
  • BRCA2 Breast Cancer Type 2 Susceptibility Protein (BRCA2) U43746 fos-related antigen (FRA1); fosLl X16707 nucleus phosphoprotein B23; Nucleophosmin (NPM);
  • Numatrin M23613 c-myc binding protein MM-1 D89667 c-fos proto-oncogene; G0S7 protein K00650 met-proto-oncogene; Hepatocyte growth factor receptor precursor (HGF-SF receptor) J02958
  • NDKA Nucleoside diphosphate kinase A
  • NDP NDP kinase A
  • Tumor metastatic process-associated protein Tumor metastatic process-associated protein
  • Matrix metalloproteinase 11 MMP11
  • Stromelysin 3 X57766 box-dependent myc-interacting protein 1 U68485 H-ras proto-oncogene
  • transforming G protein V00574 protein tyrosine phosphatase PTEN mutates in various advanced cancers 1 (MMAC1)
  • GRP 78 Immunoglobulin heavy chain binding protein (BIP) M19645
  • Interleukin-10 precursor IL-10
  • Cytokine Synthesis Hem Factor M57627
  • TX Thioredoxin
  • ADF ATL-derived factor
  • SASP Surface Associated Sulphydryl Protein
  • ENOl surface Associated Sulphydryl Protein
  • NNE non-neural enolase
  • PPH Phosphopyruvate hydratase
  • Biliverdin reductase A precursor BLVRA; BVR
  • TAT U34877 tyrosine aminotransferase
  • I-tyrosine 2-oxoglutarate aminotransferase X52520
  • Phosphoglyceride kinase 1 (PGK1; PGKA); primer
  • Detection protein 2 PRP2
  • G6PD glucose-6-phosphate dehydrogenase
  • X03674 mitochondrial phosphoenolpyruvate carboxykinase-2
  • GTA Galactosyltransferase-associated protein kinase p58
  • Subunit 1 D13900 peroxisomal bifunctional enzyme L07077 peroxisomal acyl-CoA oxidase branched subunit (BRCOX) X95190
  • Cytochrome P450 XVIIA1 (CYP17A1) M14564 peroxisomal 3-ketoacyl-CoA thiolase precursor
  • PHA2 Lung group IB phospholipase A2 precursor
  • DHFR Dihydrofolate reductase
  • Thymidylate synthase (TYMS; TS) X02308 cytosolic thymidine kinase (TK1) K02581
  • UDP-glucuronosyltransferase-2B15 precursor UDP-glucuronosyltransferase-2B15 precursor
  • UGT2B15 + microsomal 2B10 precursor UGT2B15 + microsomal 2B10 precursor (UDPGT);
  • GLCLC GLCLC, GLCL (glutamate-cysteine ligase catalytic subunit, gamma-glutamylcysteine synthetase) M90656 gamma-glutamyl hydrolase precursor (GGH; GH); Folyl polygammaglutamyl hydrolase; gamma-glu-X carboxypeptidase; Conjugase U55206 3 '-phosphoadenosine-5' -phosphosulfate synthase 1
  • PAPS synthase 1 PAPSS1
  • PAPS synthetase 1 Sulfurylase Kinase 1 (SKI) Y10387 soluble glutamate oxaloacetate transaminase 1 (GOT1); cytoplasmic aspartate aminotransferase 1;
  • acyl-CoEnzyme-A-Oxidase S69189 "very-long-chain" -specific acyl-CoA-dehydrogenase precursor (VLCAD) D43682 glutamate-cysteine ligase regulatory subunit
  • Cytochrome P450 VA1 (CYP5A1) M80647 mitochondrial aldehyde dehydrogenase precursor (class 2); ALDHI; ALDH2 Y00109
  • DHICA 5, 6-dihydroxyindole-2-carboxylic acid oxidase precursor
  • TRP-1 Tyrosinase-related protein 1
  • Catalase B Glycoprotein-75 (GP75) X51420
  • TN Tenascin precursor
  • HXB Hexabrachion
  • cytotactin cytotactin
  • Neuronectin cytonectin
  • GMEM miotic endogenous antigen
  • Matrix Metalloproteinase 15 Z48482
  • Matrix Metalloproteinase 14 MMP14
  • D26512 Matrix Metalloproteinase 1 (MMP1) X54925 Vinculin M33308 Vimentin (VIM) X56134; M14144
  • Serum amyloid Al precursor SAA1 M23698 senescence marker protein 30 (SMP30); Regucalcin (RGN; RC) D31815
  • Ubiquitin "cross-reactive" protein precursor UCRP
  • alpha-inducible interferon Interferon-induced 17kDa protein
  • G1P2 G1P2
  • ISG15 M13755
  • LAMC2 Laminin gamma-2 subunit precursor
  • PAF1 peroxisome assembly factor 1
  • PXMP3 Peroxisomal membrane protein 3
  • PMP3 Peroxisomal membrane protein 3
  • PMP35 35kDa peroxisomal membrane protein
  • PX1 Peroxisome Biogenesis Disorder Protein 1
  • GAT2 mitochondrial glutamate oxaloacetate transaminase 2
  • Aspartate aminotransferase 2 Transaminase A M22632 nck, ash & phospholipase C gamma binding
  • NAP4 Protein
  • XPF Xeroderma Pigmentosum Group F Complementary Protein
  • ERCC4 DNA excision repair protein
  • Replication factor A protein 4 (RPA4; RFA) U24186 utY homolog (hMYH) U63329 beta Crystallin A4 (CRYBA4) U59057
  • Heat shock protein beta-3 (HSPB3); Heat shock 17kDa protein; HSPL27 U15590 probable protein disulfide isomerase P5 precursor D49489
  • HSP90 90kDa heat shock protein beta
  • HSP84 84kDa heat shock protein beta
  • HSPCB M16660 microsomal UDP-glucuronosyltransferase-1-6 precursor (UDPGT; UGT1.6; UGT1F; GNT1) J04093
  • Glutathione-S-transferase mu 3 (GSTM3); GST5 J05459
  • Cytochrome P450 1A1 (CYP1A1); P450-P1; P450 form 6; P450-C K03191
  • PPAR-alpha Peroxisome proliferator-activated receptor alpha
  • PDIR Protein Disulfide Isomerase Related Protein Precursor
  • Acyl-coenzyme A cholesterol acyltransferase (ACAT); Monocyte / macrophage serine esterase (hMSE); CES2 L07765 serum paraoxonase / aryl esterase 3 (PON3); Serum aryldiacylphosphatase 3; aromatic esterase 3 (A-esterase 3) L48516 cytochrome P450 XXIB (CYP21B); Steroid 21-hydroxylase; CYP21A2 M12792; M23280
  • Cytochrome P450 IID6 (CYP2D6); P450 DB1; Debrisoquine-4-hydroxylase M20403 microsomal UDP-glucuronosyltransferase 1-1 precursor (UDPGT; UGT1.1; UGT1A; GNTl); Bilirubin-specific isozyme 1 (hUG-BRl) M57899 microsomal UDP-glucuronosyltransferase-1-4-
  • HHSF4 Heat shock transcription factor 4 D87673 extracellular superoxide dismutase precursor (EC-SOD; SOD3) J02947
  • DNAJ protein homolog 2 (DNAJ2; hDJ2; HSJ2) D13388
  • DUP Downstream protein
  • MRP1 Mismatch Repair Protein 1
  • Heat shock 70kDa protein 4 HSPA4
  • HSP70RY HSPA4
  • CCT-theta CCTQ; CCT8; KIAA0002 D13627 mitochondrial stress 70 protein precursor;
  • GRP75 75kDa glucose controlled protein
  • PBP74 Peptide binding protein 74
  • MOT Mortalin
  • FLAP endonuclease 1 FLAP endonuclease 1 (FEN1); Maturation factor 1
  • MF1 L37374 FK506 binding protein 12 (FKBP12); Peptidyl prolyl cis trans isomerase (PPIase); Rotamase M34539; M80199;
  • HSF2 Heat shock factor protein 2
  • HSTF2 Heat shock transcription factor 2
  • ADPG 3-methyladenine DNA glycosylase
  • Calreticulin precursor CRP55
  • Calregulin HACBP
  • ERp60 52-kDa ribonucleoprotein autoantigen
  • Transformation-sensitive protein IEF SSP 3521.
  • Heat shock protein beta 2 (HSPB2); DMPK binding protein; MKBP S67070 alpha Crystallin A chain (CRYAA; CRYA1) U05569
  • Nicotinamide N-methyltransferase U08021 phenol sulfating phenol sulfotransferase 1 (PPST1); thermostable phenol sulfotransferase (TS-PST); HAST1 / HAST2; ST1A3; STP1 + PPST2; ST1A2; STP2 + monoamine sulfating phenol sulfotransferase U09031 + U28170 + L19956
  • DPD dihydropyrimidine dehydrogenase precursor
  • DPD dihydrouracil dehydrogenase
  • Dihydrothymin-N dihydropyrimidine dehydrogenase precursor
  • DTYD Dehydrogenase U09178 transcriptional regulator atrX; "X-Linked” - Helicase II (XH2); "X-linked” core protein (XNP);
  • PSMD2 proteasome regulatory subunit S2
  • TRIP2 Tumor Necrosis Factor Type 1 Receptor Associated Protein
  • DDBA pl27 55.11 protein U12596 damage-specific DNA binding protein pl27 subunit
  • T complex protein 1 delta subunit TCPl delta
  • CCT delta CCT delta
  • SRB Stimulator of RNA-binding tar
  • T complex protein 1 eta subunit TCPl eta
  • CCT-eta CCT-7
  • PBGD Porphobilinogen deaminase
  • HMBS Hydroxymethylbilane synthase
  • SOD2 superoxide dismutase-2 precursor
  • GRP94 94kDa glucose controlled protein
  • TRA1 Antigen 1
  • UNG2 uracil DNA glycosylase 2
  • T-complex protein 1-alpha subunit TCPl-alpha
  • T complex protein 1 gamma subunit TCPl gamma
  • Transcription factor IIH (TFIIH); 52-kDa "basic" - transcription factor 2 subunit (BTF2p52) Y07595
  • XRCC2 x-ray repair cross-complementing protein 2
  • Ubiqitin-conjugating enzyme E2-17-kDa (UBE2B);
  • Ubiquitin-protein ligase Ubiquitin-protein ligase
  • Ubiquitin carrier protein Ubiquitin carrier protein
  • Heat shock protein 40 homolog (HSP40 homolog); DNAJW U40992
  • FKBP51 51kDa-FK506 binding protein
  • Prolyl cis trans isomerase PPIase
  • rotamase PPIase
  • FKBP54 FFI antigen
  • HSP90 binding immunophilin U42031 hematopoietic progenitor kinase (HPK1) U66464
  • a set of genes is preferably examined for methylation, in which up to 25% of the tab. 1 genes listed are not included.
  • the chemically pretreated DNA sequence of the genes to be detected is preferably at least 95% correct with the correspondingly pretreated DNA sequence of the genes from Tab. 1 match. Sequences that are 100% identical in a section of the exon that is at least 25 base pairs long are referred to as homologous in this invention. This also applies to sequences whose homology can only be recognized by taking possible frame shifts into account.
  • the genomic sequences of the genes to be examined can be derived by comparing the respective cDNA sequences with publicly accessible databases in which genomic sequences are stored.
  • Example 1 Cultivation of HT-29 P208 cells, cell harvest and preparation of chromosomal DNA
  • HT-29 P208 cells (5x104 cells / ml) were seeded in culture dishes (33x5 cm) and grown for 5 days in DMEM / Ham's F-12 medium supplemented with 10% fetal bovine serum at 37 ° C and 5% CO 2 up to a 95% confluence (Campbeil-Thompson, M. and Bhardwaj, B., Cancer Research 2001, 61, 632-640). The cells were then incubated for 24 h in medium without bovine serum.
  • the cells were, in 3 parallel cultures for 6 h and 24 h, either with medium, medium supplemented with 10 ng / ml TGF-bl, medium supplemented with 10ng / ml IL-lb, medium supplemented with trichostatin (50 nM) and Medium supplemented with Milrinone (50 ⁇ M) incubated.
  • the medium-free cells were treated with trypsin, centrifuged and resuspended in 200 ⁇ l PBS buffer (Fritsch and Maniatis eds., Molecular Cloning: A Laboratory Manual, 1989) and at -20 ° C. stored.
  • the chromosomal DNA was purified using a QIAamp kit according to the manufacturer's instructions (Qiagen, Hilden).
  • the DNA samples (20 ng) were digested with the restriction enzyme Mssl.
  • the digested DNA was chemically converted with hydrogen sulfite (bisulfite, disulfite) and a radical scavenger at elevated temperature (DE 10050942).
  • hydrogen sulfite bisulfite, disulfite
  • a radical scavenger at elevated temperature (DE 10050942).
  • the chemically pretreated DNA was then amplified in a polymerase chain reaction using a heat resistant DNA polymerase.
  • the multiplex PCR reactions were carried out with a thermal cycler (Eppendorf GmbH) using 10 ng of bisulfite-treated DNA, 6 pmol each of primer oligonucleotides (mixture of up to 32 individual primer oligonucleotides, see Table 3), each of 800 ⁇ M dNTP and 4.5 mM magnesium chloride.
  • the cycler program was as follows: Step 1, 14 min at 96 oC; Step 2, 60 sec 96 oC; Step 3, 45 sec 55 oC; Step 4, 75 sec 72 oC; Step 5, 10 min at 72 oC; steps 2 to 4 were repeated 39 times.
  • the DNA fragments of 64 different genes listed in Table 3 were amplified with 6 sets of multiplex PCR (mPCR) and bisulfite-treated DNA as a template, as described above.
  • mPCR multiplex PCR
  • the mPCR reactions (I, J, K, L, M, N) of the genomic, bisulphite-treated DNA were compared with the combination of Primer oligonucleotides performed as listed in Table 3.
  • primer pairs listed in Table 1 are particularly preferred.
  • Example 3 Determination of the methylation status of selected genes
  • the amplificates produced in Example 2 were hybridized with 512 oligonucleotides which were bound to a solid phase (Model, F. and Adorjan, P., Bioinformatics. 2001, 17 Suppl. 1, 157-164).
  • the solid phase loaded with oligonucleotides is referred to below as the oligonucleotide array.
  • the detectability of the hybridization product is based on Cy5 fluorescence-labeled primer oligonucleotides that were used for the amplification.
  • a hybridization reaction of the amplified DNA with the oligonucleotide for example
  • GTTTTTTTCGTTTTAGAG (sequence ID 6) only takes place if a methylated cytosine is present at the said site of the bisulfite-treated DNA.
  • the methylation status of the specific cytosine can thus be determined via the hybridization product.
  • This oligonucleotide is identical to the oligonucleotide which was previously used to analyze the methylated status of the sample, with the exception that the oligonucleotide carries a thymine base at the position to be analyzed instead of the cytosine base, for example GTTTTTTTTGTTTTAGAG (sequence ID 7).
  • a hybridization reaction therefore only takes place if one is not methylated cytosine is present at the position to be analyzed.
  • the fluorescence signals were detected by scanning the oligonucleotide arrays using the Genpix 4000A fluorescence scanner (Axon Instruments, USA). The fluorescence signals were quantified using the Genepix 3.0 analysis software (Axon Instruments, USA).
  • the DNA methylation patterns of HT29-P208 cells grown with cells treated with IL-Ib (interleukin) or TGF-bl (transforming growth factor) were determined.
  • the results obtained were stored in databases and the CpG dinucleotides with different methylation status were identified.
  • the methylation patterns were compared using cluster analyzes and statistical methods (Model, F. and Adorjan, P., Bioinformatics. 2001, 17 Suppl. 1, 157-164).
  • Example 4 Change in the methylation status in HT29 cells by exogenous cytokines and low-molecular-weight active substances.
  • Each of these detection oligonucleotides was designed to hybridize to bisulfite converted sequences located at CpG sites that were either unmethylated (TG) or methylated (CG) in their original state.
  • the hybridization conditions were selected to allow the detection of Show differences in single nucleotides of the variants TG and CG.
  • the ratios of the two signals were calculated based on the comparison of the intensities of the fluorescent signals.
  • the information is then determined in a weighted matrix (see FIG. 1, 2) with regard to the CpG methylation difference between two classes, treated and untreated HT29-P208 cells.
  • the p-weighted methylation (p-value ⁇ 0.05, F. Model, P. Adorjan, A. Olek, C. Piepenbrock, Feature selection for DNA methylation based cancer classification. Bioinformatics. 2001 Jun; 17 Suppl l: S157 -64) shows a clear distinction between the two groups, which can be seen from the different gray shading. A higher degree of methylation correlates with a darker gray value.
  • TGF-bl and IL-lb change the methylation status of different genes.
  • Genes that code for enzymes that catalyze the biotransformation of toxicological substances are particularly preferred in the analysis of changed methylation patterns, which in turn reflect changes in gene expression.
  • the genes of the cytochrome P450 family play a central role here. Animal experiments have shown, among other things, that one of the genes from this class, Cyplal, was induced after exposure of mice to beta-naphthoflavone (Arch Biochem Biophys 2000 Apr 1; 376 (1): 66-73).
  • the genomic DNA sequence encoding the cyplal gene must be identified. For this purpose, for example, the cDNA sequence (Genbank Acc.
  • NM_000499 can be compared with a genomic database (eg Genbank htgs), usually using the BLAST comparison algorithm, which is available on the Internet (www.ncbi.nlm.nih.gov), is used.
  • a genomic database eg Genbank htgs
  • the section of the genomic DNA that encodes Cyplal can be identified (Genbank Acc.AC020705).
  • Genomic sections in the area of the promoter and the first exon or intron are preferably examined for methylation differences, since relevant CpG dinucleotides, the methylation of which influences gene expression, are preferably found in these sections.
  • exon 1 (underlined) was located in the following genomic sequence section:
  • TATGTTAAATGGTATTGG and CATCCAAAAACTAT can be used, with which defined fragments with a length of 1316 base pairs can be amplified. These amplificates serve as probes which are hybridized to oligonucleotides previously bound to a solid phase, for example CTACCCCGTAATA, the cytosine to be detected being in position 837 of the amplificate is located.
  • the detection of the hybridization product is based on Cy3 or Cy5 fluorescence-labeled primer oligonucleotides that were used for the amplification.
  • a hybridization reaction of the amplified DNA with the oligonucleotide only occurs if there is a methylated cytosine in the bisulfite-treated DNA at this point. The methylation status of the respective cytosine to be examined thus decides on the hybridization product.
  • Example 6 Classification of a chemical substance into a toxicity class by determining the methylation pattern
  • the DNA methylation pattern of a group of exposed and a group of non-exposed organisms, for example experimental animals must first be examined. The results are stored in a database and the CpG dinucleotides identified, which are methylated differently between the two groups. Then the methylation pattern of the substance to be assessed is compared with known methylation patterns of other chemical substances. Information on the properties of the substance to be investigated can be obtained from the toxicological properties of those chemical substances with a similar methylation pattern.
  • the present invention also relates to a kit consisting of a reagent containing bisulfite, a set of primer oligonucleotides comprising at least two oligonucleotides, the sequences of which each correspond to at least one 18 base pair long section of the base sequences of the genes to be examined or complement them.
  • a kit consisting of a reagent containing bisulfite, a set of primer oligonucleotides comprising at least two oligonucleotides, the sequences of which each correspond to at least one 18 base pair long section of the base sequences of the genes to be examined or complement them.
  • tär for the production of the amplificates, oligonucleotides and / or PNA oligomers, a control nucleic acid and instructions for performing and evaluating the method described.
  • Weighted matrix of the methylation status (fluorescence signal CG-Oligo x (fluorescence signal CG-Oligo + fluorescence signal TG-Oligo) -1) of 40 CpGs in untreated HT29-P208 cells (A) and TGF-bl treated HT29-P208 cells (B) .
  • the numbers 1-3 indicate 3 independent experiments (cell treatments and methylation analysis).
  • Each horizontal bar represents a CpG whose
  • Methylation status with a significance of p ⁇ 0.05, is different in the two analysis groups. A higher degree of methylation corresponds to the darker shade of gray, a lower degree of methylation corresponds to the lighter shade of gray.
  • HT29-P208 cells A
  • IL-Ib treated HT29-P208 cells B
  • the numbers 1-3 indicate 3 independent experiments (cell treatments and methylation analysis).
  • Each horizontal bar represents a CpG whose methylation status, with a significance of p ⁇ 0.05, is different in the two analysis groups.
  • a higher degree of methylation corresponds to the darker shade of gray, a lower degree of methylation corresponds to the lighter shade of gray.
  • CpGs which are represented by the indicated oligo-SEQ IDs, were examined from the following genes: TGF-a (AI, oligo SEQ IDs 6, 7; A2, oligo SEQ IDs 8, 9), EGFR (B1, oligo SEQ IDs 20 , 21; B2, oligo SEQ IDs 22, 23), ANTl (Cl, oligo SEQ IDs 32, 33; C2, oligo SEQ IDs 34, 35) and E-Cadherin (Dl, oligo SEQ IDs 13, 14; D2, oligo SEQ IDs 15, 16).
  • the numerical values of the y-axis show the methylation status, calculated as the quotient of the fluorescence signal of the CG oligo over the sum of the fluorescence signals of the TG and CG oligo.
  • CpGs which are represented by the specified oligo-SEQ IDs, were examined from the following genes: EGFR (AI, oligo SEQ IDs 22, 23), ANTl (B1, oligo

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Abstract

La présente invention concerne un procédé permettant le diagnostic toxicologique. Un échantillon d'ADN est prélevé chez un être vivant ou une culture cellulaire qui a été préalablement exposé à une substance donnée à activité toxicologique à analyser. L'ADN contenu dans cet échantillon est prétraité chimiquement et la séquence de bases d'une partie de l'ADN modifié est déterminée. A partir de cela, un état de méthylation caractéristique de l'échantillon ou un motif de méthylation caractéristique est déterminé. Grâce à la comparaison avec des données relatives à des états de méthylation d'autres échantillons, l'activité d'une substance sur l'être vivant ou sur la culture cellulaire est déterminée et/ou comparée avec celle d'autres substances d'un point de vue toxicologique.
EP01996625A 2000-11-14 2001-11-08 Procede de detection d'etats de methylation afin de permettre le diagnostic toxicologique Withdrawn EP1337668A2 (fr)

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DE10056802 2000-11-14
DE10056802A DE10056802B4 (de) 2000-11-14 2000-11-14 Verfahren zur Detektion von Methylierungszuständen zur toxikologischen Diagnostik
PCT/EP2001/012951 WO2002040710A2 (fr) 2000-11-14 2001-11-08 Procede de detection d'etats de methylation afin de permettre le diagnostic toxicologique

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