EP1332228A2 - Diagnostic de maladies associees au gene cdk4 en determinant le degre de methylation du gene cdk4 - Google Patents

Diagnostic de maladies associees au gene cdk4 en determinant le degre de methylation du gene cdk4

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Publication number
EP1332228A2
EP1332228A2 EP01992793A EP01992793A EP1332228A2 EP 1332228 A2 EP1332228 A2 EP 1332228A2 EP 01992793 A EP01992793 A EP 01992793A EP 01992793 A EP01992793 A EP 01992793A EP 1332228 A2 EP1332228 A2 EP 1332228A2
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Prior art keywords
seq
dna
oligomer
cancer
cdk4
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German (de)
English (en)
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Alexander Olek
Christian Piepenbrock
Kurt Berlin
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Epigenomics AG
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Epigenomics AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • C12Q2600/154—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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to nucleic acids, oligonucleotides, PNA oligomers and a method for diagnosing diseases which are related to the genetic and / or epigenetic parameters of the gene Cdk4 and in particular its methylation status.
  • cyclin Dl An important factor in the Gl phase of the cell cycle is cyclin Dl. It has been shown that cyclin Dl can produce lymphoid tumors in cooperation with activated myc genes in transgenic mice (Lovec H, Grzeschiczek A, Kowalski MB, Moroy T. Cyclin Dl / bcl-1 co-rates with myc genes in the generation of B-cell lymphoma in transgenic mice.EMBO J. 1994 Aug l; 13 (15): 3487-95). Cyclin Dl can thus act as an oncogene, but requires cell-specific cooperating partners. Furthermore it could be shown that not only cyclin Dl has oncogenic potential, but also the cyclin-dependent cyclin-dependent one
  • Cdk4 is associated with acute lymphoblastic leukemia (Mekki Y, Catallo R, Bertrand Y, Manel AM, Ffrench P, Baghdassarian N, Duhaut P, Bryon PA, Ffrench M.
  • Enhanced expression of pl6ink4a is associated with a poor prognosis in childhood acute lymphoblastic leukemia. Leukemia. 1999 Feb; 13 (2): 181-9; Omura-Minamisawa M, Diccianni MB, Batova A, Chang RC, Bridgeman LJ, Yu J, Pullen J, Bowman WP, Yu AL.
  • pl ⁇ and Cdk4 in oral premalignant lesions and oral squamous cell carcinomas are semi-quantitative immunohistochemical studies. J Oral Pathol Med. 1999 Apr; 28 (4): 158-64), on non-small cell lung cancer (Malusecka E, Zborek A, Krzyzowska-Gruca S. Changes in expression of pRb, pl ⁇ and cyclin Dl in non-small cell lung cancer: an immunohistochemical study. Folia Histochem Cytobiol.
  • Cdk4 appears to be responsible for the development of the malignant peripheral nerve sheath tumor (Berner JM, Sorlie T, Mertens F, Henriksen J, Saeter G, Mandahl N, Brogger A, Myklebost O, Lothe RA.
  • Chromosome band 9p21 is frequently altered in malignant peripheral nerve sheath tumors: studies of CDKN2A and other genes of the pRB pathway. Genes Chromosomes Cancer. 1999 Oct; 26 (2): 151-60) and prostate cancer (Lee CT, Capodieci P, Osman I, Fazzari M, Ferrara J , Scher HI, Cordon-Cardo C. Overexpression of the cyclin-dependent kinase inhibitor pl ⁇ is associated with tumor recurrence in human prostate cancer. Clin Cancer Res. 1999 May; 5 (5): 977-83); also kidney disease (Wolf G. Angiotensin II is involved in the progression of renal disease: im- portance of non-hemodynamic mechanisms. Nephrology.
  • Cdk4 Other associations of Cdk4 concern diffuse large B-cell lymphoma (Rao PH, Houldsworth J, Dyomina K, ParsaNZ, Cigudosa JC, Louie DC, Popplewell L, Off ⁇ t K, Jhanwar SC, Chaganti RS.Cromosomal and gene amplification in diffuse large B -cell lymphoma. Blood.
  • heritable melanoma and ⁇ ävi Greene MH The genetics of hereditary melanoma and nevi. 1998 update. Cancer. 1999 Dec 1; 86 (11 Suppl): 2464-77).
  • 5-Methylcytosine is the most common covalently modified base in the DNA of eukaryotic cells. For example, it plays a role in the regulation of transcription, in genetic imprinting and in tumorigenesis. The identification of 5-methylcytosine as a component of genetic information is therefore of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing since 5-methylcytosine has the same base pairing behavior as cytosine. In addition, in the case of PCR amplification, the epigenetic information which the 5-methylcytosines carry is completely lost.
  • a relatively new and the most frequently used method for the investigation of DNA for 5-methylcytosine is based on the specific reaction of bisulfite with cytosine, which is converted into uracil after alkaline hydrolysis, which corresponds to the thymidine in its base pairing behavior.
  • 5-methylcytosine is not modified under these conditions.
  • the original DNA is thus converted in such a way that methylcytosine, which originally cannot be distinguished from the cytosine by its hybridization behavior, can now be detected by "normal" molecular biological techniques as the only remaining cytosine, for example by amplification and hybridization or sequencing.
  • the bisulfite technique has so far been used with a few exceptions (e.g. Zeschnigk M, Lieh C, Buiting K, Doerfler W, Horsthemke B. A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hum Genet. 1997 Mar-Apr; 5 (2): 94-8) used only in research. However, short, specific pieces of a known gene are always amplified after bisulfite treatment and either completely sequenced (Olek A, Walter J. The pre-implantation ontogeny of the H19 methylation imprint. Nat Genet.
  • Genomic sequencing indicates a correlation between DNA hypomethylation in the 5 'region of the pS2 gene and its expression in human breast cancer cell lines. Genes. 1995 May 19; 157 (1-2): 261-4; WO 97 46705, WO 95 15373 and WO 97 45560.
  • 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 (Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988 Oct 15; 60 (20): 2299-301).
  • An analyte is embedded in a light-absorbing matrix. The matrix is evaporated by a short laser pulse and the analyte molecule so 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.
  • MALDI-TOF spectrometry is ideal for the analysis of peptides and proteins.
  • the analysis of nucleic acids is somewhat more difficult (Gut I G, Beck S. DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry. Current Innovations and Future Trends. 1995, 1; 147-57).
  • the sensitivity for nucleic acids is about 100 times worse than for peptides and decreases disproportionately with increasing fragment size. For nucleic acids that have a backbone that is often negatively charged, the ionization process through the matrix is much more inefficient.
  • MALDI-TOF spectrometry the choice of the matrix plays an eminently important role.
  • 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 oligonucleotides and / or PNA oligomers for the detection of cytosine methylations and a method which is suitable for the diagnosis of The genetic and epigenetic parameters of the Cdk4 gene are particularly suitable.
  • the invention is based on the finding that cytosine methylation patterns in particular are particularly suitable for diagnosing diseases associated with Cdk4.
  • the invention is based on the knowledge that genetic and epigenetic parameters and in particular the cytosine methylation pattern of the Cdk4 gene are particularly suitable for diagnosing diseases associated with Cdk4.
  • nucleic acid comprising an at least 18 base long sequence section of the chemically pretreated DNA of the gene Cdk4 according to one of the Seq. ID No.l to Seq. ID No.4 solved.
  • the chemically modified nucleic acid has so far not been associated with the determination of genetic and epigenetic parameters.
  • an oligonucleotide or oligomer for detecting the cytosine methylation state in chemically pretreated DNA comprising at least one base sequence with a length of at least 13 nucleotides, which is linked to a chemically pretreated DNA of the gene Cdk4 according to one of the Seq. ID No.l to Seq. ID No.4 hybridized.
  • the oligomer probes according to the invention represent important and effective tools which make it possible to determine the genetic and epigenetic parameters of the Cdk4 gene in the first place.
  • the base sequence of the oligomers preferably comprises at least one CpG dinucleotide.
  • the probes can also be in the form of a PNA (Peptide Nucleic Acid), which has particularly preferred pairing properties.
  • PNA Peptide Nucleic Acid
  • Particularly preferred are oligonucleotides according to the invention in which the cytosine of the CpG dinucleotide is the 5th to 9th nucleotide from the 5 'end of the 13 mer, in the case of PNA oligomers it is preferred that the cytosine of the CpG dinucleotide is the 4th - 6. Nucleotide from the 5 'end of the 9 mer.
  • the oligomers according to the invention are normally used in so-called sets which contain one of the sequences of Seq for each of the CpG dinucleotides. ID No.l to Seq. ID No.4 comprise at least one oligomer. A set is preferred which comprises at least one oligomer for each of the CpG dinucleotides from one of Seq ID No. 1
  • the invention provides a set of at least two oligonucleotides, which act as so-called primer oligonucleotides for the amplification of DNA sequences of one of the Seq. ID No.l to Seq. ID No.4 or sections thereof can be used.
  • At least one oligonucleotide is bound to a solid phase.
  • the present invention further relates to a set of at least 10 oligomers (oligonucleotides and / or PNA oligomers) which are used to detect the cytosine methylation state in chemically pretreated genomic DNA (Seq. ID No. 1 to Seq. ID No.4). With these probes the diagnosis of genetic and epigenetic parameters of the Cdk4 gene is possible.
  • the set of oligomers can also be used to detect single nucleotide polymorphisms (SNPs) in the chemically pretreated DNA of the Cdk4 gene according to one of the Seq. ID No.l to Seq. ID No.4 can be used.
  • an arrangement made of different oligonucleotides and / or PNA oligomers (a so-called "array") provided by the invention is also bound to a solid phase.
  • This array of different oligonucleotide and / or PNA oligomer sequences can be characterized in that it is arranged on the 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.
  • nitrocellulose and plastics such as nylon are also possible, which can be in the form of spheres or as resin matrices.
  • the invention therefore furthermore relates to a method for producing an array fixed on a carrier material for analysis in connection with Cdk4-associated diseases, in which at least one oligomer according to the invention is attached to a solid phase is coupled.
  • Methods for producing such arrays are, for example, from the US
  • the invention further relates to a DNA chip for analysis in connection with Cdk4-associated diseases, which comprises at least one nucleic acid according to the present invention.
  • DNA chips are known, for example, from US Pat. No. 5,837,832.
  • the present invention also relates to a kit which, for example, consists of a reagent containing bisulfite, a set of primer oligonucleotides comprising at least two oligonucleotides, the sequences of which each have at least an 18 base pair section of the base sequences listed in the appendix (Seq. ID No. 1 to Seq . ID No.4) correspond or are complementary to them, oligonucleotides and / or PNA oligomers as well as instructions for carrying out and evaluating the described method can exist.
  • a kit in the sense of the invention can also contain only parts of the aforementioned components.
  • the invention further provides a method for determining genetic and / or epigenetic parameters of the Cdk4 gene by analyzing cytosine methylations and single nucleotide polymorphisms, which comprises the following steps:
  • a genomic DNA sample is chemically treated in such a way that at the 5 'position unmethylated cytosine bases are converted into uracil, thymine or another base which is unlike cytosine in terms of hybridization behavior. This is understood below as chemical pretreatment.
  • the genomic DNA to be analyzed is preferably obtained from the usual sources for DNA, such as cells or cell components, for example cell lines, biopsins, blood, sputum, stool, urine, brain-spinal fluid, tissue embedded in paraffin, for example tissue from eyes, Intestine, kidney, brain, heart, prostate, lung, breast or liver, histological slides or combinations thereof.
  • sources for DNA such as cells or cell components, for example cell lines, biopsins, blood, sputum, stool, urine, brain-spinal fluid, tissue embedded in paraffin, for example tissue from eyes, Intestine, kidney, brain, heart, prostate, lung, breast or liver, histological slides or combinations thereof.
  • the treatment of genomic DNA with bisulfite (hydrogen sulfite, disulfite) and subsequent alkaline hydrolysis, which leads to conversion, is preferably used.
  • Fragments are amplified from this chemically pretreated genomic DNA using sets of primer oligonucleotides according to the invention and a preferably heat-stable polymerase. For statistical and practical considerations, more than ten different fragments that are 100-2000 base pairs long are preferably amplified.
  • the amplification of several DNA sections can be carried out simultaneously in one and the same reaction vessel. The amplification is usually carried out by means of the polymerase chain reaction (PCR).
  • 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 base sequences listed in the appendix (Seq. ID No. 1 to Seq. ID No.4) that is at least 18 base pairs long ,
  • the primer oligonucleotides are preferably characterized in that they contain no CpG dinucleotide.
  • At least one primer oligonucleotide is bound to a solid phase during the amplification.
  • the different oligonucleotide and / or PNA oligomer sequences can be arranged on a flat solid phase in the form of a rectangular or hexagonal grid, the solid phase surface preferably consisting of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold, other materials such as nitrocellulose or plastics can also be used.
  • the fragments obtained by means of the amplification can carry a directly or indirectly detectable label. Markings in the form of fluorescent markings, radionuclides or detachable molecular fragments with typical mass, which can be detected in a mass spectrometer, are preferred, it being preferred that the fragments produced have a single positive or negative net charge for better detectability in the mass spectrometer.
  • the detection can be performed and visualized using matrix assisted laser desorption / ionization mass spectrometry (MALDI) or using electrospray mass spectrometry (ESI).
  • MALDI matrix assisted laser desorption / ionization mass spectrometry
  • ESI electrospray mass spectrometry
  • the amplificates obtained in the second process step are then hybridized to a set of oligonucleotides and / or PNA probes or to an array.
  • the hybridization is carried out in the manner given below.
  • the set used in the hybridization preferably consists of at least 10 oligonucleotide or PNA oligomer probes.
  • the amplificates serve as probes that hybridize to oligonucleotides previously bound to a solid phase.
  • 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 listed in the appendix, which contains at least one CpG dinucleotide.
  • the cytosine of the CpG dinucleotide is the 5th to 9th nucleotide viewed from the 5 'end of the 13 mer.
  • 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 listed in the appendix, 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 9mer.
  • the non-hybridized amplificates are removed.
  • the hybridized amplificates are detected. It is preferred that labels attached to the amplificates can be identified at any position on the solid phase at which an oligonucleotide sequence is located.
  • the labels of the amplified products are fluorescent labels, radionuclides or detachable molecular fragments with typical mass, which can be detected in a mass spectrometer.
  • the detection of the amplified products, fragments of the amplified products or probes complementary to the amplified products in the mass spectrometer is preferred, the detection using matrix assisted laser desorption / ionization mass spectrometry (MALDI) or using electrospray mass spectrometry (ESI) being able to be carried out and visualized.
  • MALDI matrix assisted laser desorption / ionization mass spectrometry
  • ESI electrospray mass spectrometry
  • the fragments generated can have a single positive or negative net charge for better detectability in the mass spectrometer.
  • the aforementioned method is preferred used to determine genetic and / or epigenetic parameters of the Cdk4 gene.
  • the oligomers or arrays thereof according to the invention and a kit according to the invention are to be used for the diagnosis of a disease associated with Cdk4 by analysis of methylation patterns of the Cdk4 gene. According to the invention, the use of the method for the diagnosis of important genetic and / or epigenetic parameters within the Cdk4 gene is preferred.
  • the method according to the invention is used, for example, to diagnose acute lymphoblastic leukemia, acute lymphoblastic leukemia of T cells, acute myeloid leukemia, uterine cancer, stomach cancer, Alzheimer's disease, precancerous changes in the oral mucosa and squamous cell carcinoma of the oral mucosa, non-small cell lung cancer, parostalemic cancer peripheral nerve sheath tumor, non-small cell lung cancer, parostal osteosarcoma, malignant peripheral nerve sheath tumor, prostate cancer, kidney disease, breast cancer, diffuse large B-cell lymphoma, multiple myeloma, round cell liposarcoma, tuberous sclerosis, ovarian cancer and veritable melanoma.
  • the nucleic acids of Seq. ID No.l to Seq. ID No.4 can be used for the diagnosis of genetic and / or epigenetic parameters of the Cdk4 gene.
  • the present invention further relates to a method for producing a diagnostic for the diagnosis of diseases associated with Cdk4 by analyzing methylation patterns of the Cdk4 gene, the diagnostic being characterized in that at least one nucleic acid, according to the present invention, optionally together is used with suitable additives and auxiliaries for its production.
  • the present invention further relates to a diagnostic agent for diseases associated with Cdk4 by analyzing methylation patterns of the Cdk4 gene, which comprises at least one nucleic acid according to the invention, optionally together with suitable additives and auxiliaries.
  • the present invention furthermore relates to the diagnosis and / or prognosis disadvantageously
  • hybridization in the sense of the present invention is to be understood as binding to form a duplex structure of an oligonucleotide to a completely complementary sequence in the sense of the Watson-Crick base pairings in the sample DNA.
  • Stringent hybridization conditions are to be understood as those conditions in which hybridization takes place at 60 ° C. in 2.5 ⁇ SSC buffer, followed by several washing steps at 37 ° C. in a lower buffer concentration and remains stable.
  • the term “functional variants” denotes all DNA sequences that are complementary to a DNA sequence that hybridize to the reference sequence under stringent conditions and have an activity similar to the corresponding polypeptide according to the invention.
  • Genetic parameters in the sense of this invention are mutations and polymorphisms of the Cdk4 gene and sequences that are still required for its regulation.
  • insertions, deletions, point mutations, inversions and polymorphisms and particularly preferably SNPs (single nucleotide polymorphisms) are to be referred to as mutations.
  • Polymorphisms can also be insertions, deletions or inversions.
  • Epigenetic parameters in the sense of this invention are, in particular, cytosine methylations and further chemical modifications of DNA bases of the Cdk4 gene and sequences that are also required for its regulation. Further epigenetic parameters are, for example, the acetylation of histones, which, however, cannot be analyzed directly with the method described, but in turn is corrected with DNA methylation.
  • FIG. 1 shows the differentiation of cell lines and samples from patients with the diagnosis ALL and cell lines and samples from patients with the diagnosis AML.
  • a high probability of methylation corresponds to dark gray signals (these appear red in the color illustration), a low probability corresponds to light gray signals (these appear green in the color illustration) and black mean values.
  • the samples on the left (A) of FIG. 1 are assigned to the group from ALL, and those on the right (B) AML.
  • Seq. ID No. 1 shows the sequence of the chemically pretreated genomic DNA of the gene Cdk4
  • Seq. ID No.2 shows the sequence of a second chemically pretreated genomic DNA of the gene Cdk4
  • Seq. ID No.3 shows the reverse complementary sequence of the Seq. ID 1 of the chemically pretreated genomic DNA of the Cdk4 gene
  • Seq. ID No.4 shows the reverse complementary sequence of the Seq. ID 2 of the chemically pretreated genomic DNA of the Cdk4 gene
  • Seq. ID No.5 shows the sequence of an oligonucleotide for the amplification of Cdk4 from Example 1
  • Seq. ID No.6 shows the sequence of a second oligonucleotide for the amplification of Cdk4 from Example 1
  • Seq. ID NoJ shows the sequence of an oligonucleotide for hybridizing the amplificate of Cdk4 from Example 1
  • Seq. ID No.8 shows the sequence of a second oligonucleotide for hybridizing the amplificate of Cdk4 from Example 1
  • Seq. ID No.9 shows the sequence of a third oligonucleotide for hybridizing the amplificate of Cdk4 from Example 1
  • Seq. ID No.10 shows the sequence of a fourth oligonucleotide for hybridizing the amplificate of Cdk4 from Example 1
  • Seq. ID No. 11 shows the sequence of an oligonucleotide for hybridizing the amplificate of Cdk4 from Example 1
  • Seq. ID No.12 shows the sequence of a fifth oligonucleotide for hybridizing the amplificate of Cdk4 from Example 1
  • Seq. ID No. 13 shows the sequence of an oligonucleotide for hybridizing the amplificate of Cdk4 from Example 1
  • Seq. ID No.14 shows the sequence of a sixth oligonucleotide for hybridizing the amplificate of Cdk4 from Example 1
  • Seq. ID No. 15 shows the sequence of a seventh oligonucleotide for hybridizing the amplificate of Cdk4 from Example 1
  • Seq. ID No. 16 shows the sequence of an eighth oligonucleotide for hybridizing the amplificate of Cdk4 from Example 1
  • Seq. ID No. 17 shows the sequence of an eighth oligonucleotide for hybridizing the amplificate of Cdk4 from Example 2
  • Seq. ID No. 18 shows the sequence of an eighth oligonucleotide for hybridizing the amplificate of Cdk4 from Example 2
  • Seq. ID No. 19 shows the sequence of an eighth oligonucleotide for hybridizing the amplificate of Cdk4 from Example 2
  • Seq. ID No.20 shows the sequence of an eighth oligonucleotide for hybridizing the amplificate of Cdk4 from Example 2
  • the following example relates to a fragment of the Cdk4 gene, in which a specific CG position is examined for its methylation status.
  • Example 1 Performing the methylation analysis in the Cdk4 gene
  • a genomic sequence is treated using bisulfite (hydrogen sulfite, disulfite) in such a way that all of the cytosines that are not methylated at the 5-position of the base are modified in such a way that a base which differs in the base-pairing behavior deforms, whereas the base in the 5-position methylated cytosines remain unchanged.
  • bisulfite in the concentration range between 0.1 and 6 M is used for the reaction, an addition takes place at the unmethylated cytosine bases.
  • a denaturing reagent or solvent and a radical scavenger must be present.
  • the treated DNA sample is diluted with water or an aqueous solution. Desulfonation of the DNA (10-30 min, 90-100 ° C.) is then preferably carried out at an alkaline pH.
  • the DNA sample is amplified in a polymerase chain reaction, preferably with a heat-resistant DNA polymerase.
  • cytosines of the CDK4 gene here from the 5'UTR. Sequences of this gene can be used to distinguish samples from patients diagnosed with ALL from healthy B / T cells. For this purpose, a defined fragment with a length of 474 bp is amplified with the specific primer oligonucleotides TTTTGGTAGTTGGTTATATG (Seq. ID No. 5) and AAAAATAACACAATAACTCA (Seq. ID No. 6). This amplificate serves as a sample which hybridizes to an oligonucleotide previously bound to a solid phase to form a duplex structure, for example GATTCCTACGACCCCATA (Seq. ID No.
  • cytosine to be detected being at position 120 of the amplificate.
  • the methylated cytosine is detected with the oligonucleotide (Seq. ID No. 7), which has a guanine at the complementary site in question, whereas the unmethylated state, which is represented by a thymine, with the oligonucleotide (Seq. ID No 8), which on the relevant complementary site has an adenine is detected.
  • oligonucleotides that can be used for hybridization include the following sequences:
  • CCCTTAAACGACCCTTCC (Seq. ID No.9) and CCCTTAAACAACCCTTCC (Seq. ID
  • CCACTTCCCGCCCTTAAA (Seq. ID No.11) and CCACTTCCCACCCTTAAA (Seq. ID
  • samples from patients diagnosed with ALL can be distinguished from samples from patients diagnosed with AML.
  • a defined fragment with a length of 474 bp is amplified with the specific primer oligonucleotides TTTTGGTAGTTGGTTATATG (Seq. ID No. 5) and AAAAATAACACAATAACTCA (Seq. ID No. 6).
  • This amplificate serves as a sample which hybridizes to an oligonucleotide previously bound to a solid phase to form a duplex structure, for example CCCTTAAACGACCCTTCC (Seq. ID No. 9) and CCCTTAAACAACCCTTCC (Seq. ID No.
  • cytosine to be detected at position 276 of the amplificate CCTTACATCGAAAATCCT (Seq. ID No. 13) and CCTTACATAGAAAATCCT (Seq. ID No. 14) with the cytosine to be detected at position 349 of the amplified product, TCCAACCACGTAAAACCC (Seq. ID No. 15) and TCCAACCACATAAAACCC (Seq. ID No. 16) the cytosine to be detected at position 433 of the amplificate.
  • the methylated cytosine is detected with the oligonucleotide (Seq. ID No.
  • the detection 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 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 2 Performing the methylation analysis in the CDK4 gene
  • a genomic sequence is treated using bisulfite (hydrogen sulfite, disulfite) in such a way that none of the methylated cytotoxin at the 5-position of the base be changed so that a different behavior with regard to the base pairing
  • Base is formed, while the cytosines methylated in the 5-position remain unchanged. If bisulfite is used for the reaction, an addition takes place on the unmethylated cytosine bases. In addition, a denaturing reagent or solvent and a radical scavenger must be present. Subsequent alkaline hydrolysis then leads to the conversion of unmethylated cytosine nucleobases into uracil. This converted DNA is used to detect methylated cytosines. In the second process step, the treated DNA sample is diluted with water or an aqueous solution. Desulfonation of the DNA is then preferably carried out.
  • the DNA sample is amplified in a polymerase chain reaction, preferably with a heat-resistant DNA polymerase.
  • the PCR reactions were carried out in a thermal cycler (Eppendorf GmbH). 10 ng DNA, 0.08 ⁇ M of each primer oligonucleotide 1, 6mM dNTPs and one unit of HotstartTaq were used for a 25 ⁇ l mixture. The other conditions were chosen according to the manufacturer's instructions.
  • denaturation was first carried out at 96 ° C. for 15 minutes, then. '.36 cycles (60 seconds at 96 ° C, 45 seconds at 52 ° C and 75 seconds at 72 ° C) and a final elongation of 10 minutes at 72 ° C. The presence of the PCR products was checked on agarose gels.
  • cytosines of the CDK4 gene are examined. Sequences of this gene can be used to distinguish cell lines and samples from patients diagnosed with ALL from cell lines and samples from patients diagnosed with ALL. For this purpose, a defined fragment with a length of 474 bp is amplified with the specific primer oligonucleotides TTTTGGTAGTTGGTTATATG (Seq. ID No. 5) and AAAAATAACACAATAACTCA (Seq. ID No. 6). This amplificate serves as a sample which hybridizes to an oligonucleotide previously bound to a solid phase to form a duplex structure, for example GGAAGGGTCGTTTAAGGG (Seq. ID No.
  • oligonucleotide (Seq. ID No. 17), which has a guanine at the complementary site in question, whereas the unmethylated state, which is represented by a thymine, with the oligonucleotide (Seq. ID No 18), which has an adenine at the complementary site in question.
  • oligonucleotides that can be used for hybridization include the following sequences: GGGTTTTACGTGGTTGGA (Seq. ID No. 19) and GGGTTTTATGTGGTTGGA (Seq. ID No. 20) with the cytosine to be detected at positi on 434 of the amplificate and on the corresponding counter strand
  • the detection of the hybridization product is based on CY5 fluorescence-labeled primer oligonucleotides that were used for the amplification. Only if there is a methylated cytosine in the bisulfite-treated DNA at this point will there be a hybridization reaction of the amplified DNA with the
  • Oligonucleotide The methylation status of the individual to be examined is therefore decisive
  • the following example describes the comparison of cell lines and samples from patients diagnosed with ALL and cell lines and samples from patients diagnosed with ALL. Fluorescence-labeled primers were used for the PCRs. All PCR products from each individual were mixed and hybridized on glass slides carrying a pair of immobilized oligonucleotides at each position. 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 detect differences in single nucleotides of the TG and CG variants. 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) with regard to the CpG methylation differences between two classes of tissues.
  • the most significant CpG positions are shown at the lower end of the matrix, the significance decreases towards the top.
  • Dark gray in the original figure: red
  • light gray in the original figure: green
  • black a medium degree.
  • Each row represents a specific CpG position in a gene and each column shows the methylation profile of different CpGs for a sample.
  • a gene identification number is shown on the left; the associated gene name can be found in Table 1.
  • Table 1 also lists the corresponding gene accession numbers.
  • the number before the colon denotes the gene name and the number after the colon the specific oligonucleotide.
  • the Fisher values of the individual CpG positions are shown on the right side of FIG. The individual sample names are listed at the bottom of the figure.
  • the methylation pattern to one of the diseases associated with Cdk4, e.g. acute lymphoblastic leukemia and acute lymphoblastic leukemia from T cells, acute myeloid leukemia, uterine cancer, gastric cancer, Alzheimer's disease, precancerous change in the oral mucosa and squamous cell carcinoma of the oral mucosa, non- small cell lung cancer, parostal osteosarcoma, malignant peripheral nerve sheath tumor, non-small cell lung cancer, parostal osteosarcoma, malignant peripheral nerve sheath tumor, prostate cancer, kidney disease, breast cancer, diffuse large cell B-cell lymphoma, multiple myeloma, round cell ovarian cancer
  • the DNA methylation pattern of a group of sick and a group of healthy people must first be examined.
  • Example 2 These tests are carried out, for example, analogously to Example 1.
  • the results obtained in this way are stored in a database and the CpG dinucleotides which are methylated differently between the two groups are identified. This can be done by determining individual CpG methylation rates.
  • B. by sequencing relatively imprecise or very precisely by a methylation-sensitive "primer extension reaction”. Simultaneous analysis of the entire methylation status is also possible, and the patterns can, for example, by means of Clustering analyzes, which can be carried out, for example, by a computer, are compared.

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Abstract

L'invention concerne la séquence génomique modifiée chimiquement du gène Cdk4, des oligonucléotides ou des oligomères PNA dirigés contre cette séquence et servant à détecter l'état de méthylation de la cytosine du gène Cdk4, ainsi qu'un procédé pour déterminer des paramètres génétiques et épigénétiques du gène Cdk4.
EP01992793A 2000-11-06 2001-11-06 Diagnostic de maladies associees au gene cdk4 en determinant le degre de methylation du gene cdk4 Withdrawn EP1332228A2 (fr)

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