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  • 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
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  • 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 to a method for the diagnosis and/or therapy of diseases which have a connection with the genetic and/or epigenetic parameters of genes associated with metastasis and, in particular, with the methylation status thereof.
• metastasis The key feature of malignant cells is their ability to invade normal healthy tissue and to be disseminated through the body to distant organs. This ability, known as metastasis, is one of the most fatal metastasis of cancer. In breast cancer for example, the extent of metastasis to the lymph nodes is a key prognostic factor of the disease. Approximately 30% of cancers are metastatic at the time of diagnosis, and a further 30-40% of the remaining case harbour occult metastases.
• Metastasis is a highly complicated pathway involving multiple proteolytic enzymes, cell adhesion, deformability, cell receptors and motility. Cancer metastasis can be described in the following steps. The initial events involve establishment of the primary tumour. These comprise prise the initial transforming event and proliferation of the transformed cells followed by evasion of the immune mechanism and establishment of a nutritional supply.
• tumour cell surface proteins such as laminin, type IV collagen, and fibronectin.
• Invasion then proceeds by enzymatic means, both proteinases (serine, cysteine, aspartic proteinases and metalloproteinases) and tumour secreted hydrolytic enzymes (e.g. glycosidase, hyaluronidase and heparanase) have been implicated.
• tumour cells The next step involves the migration of tumour cells from the primary tumour.
• the movement of the cells through biological barriers may be driven by a number of factors. These include tumour-derived chemotactic factors, host-derived chemoattractants, and combinations of the two.
• tumour-derived chemotactic factors include tumour-derived chemotactic factors, host-derived chemoattractants, and combinations of the two.
• growth factors include growth factors, collagen, peptides, matrix components and proteolytic fragments of matrix components, adhesion proteins such as laminin and fibronectin, and tumour-derived attractants.
• adhesion proteins such as laminin and fibronectin
• tumour-derived attractants the importance of autocrine growth factors for transformed cell motility has also been demonstrated.
• the mobilised cells then attempt to penetrate blood vessel walls. Once the mobilised cells enter the blood stream they are embolized to distant organs. The cells may then be arrested in the lumen of small blood vessels or lymphatics. The cancer cells then proceed to extrude themselves through the walls of the vessels. Establishment of secondary tumours then proceeds by proliferation of the transformed cells followed by evasion of the immune mechanism and establishment of a nutritional supply.
• Hodgkin's disease Garcia J F et al “Loss of p16 protein expression associated with methylation of the p16INK4A gene is a frequent finding in Hodgkin's disease” Lab invest 1999 December; 79 (12):1453-9)
• Methylation based therapies could have considerable advantages over current methods of treatment, such as chemotherapy, surgery and radiotherapy. They may even provide a means of treating tumors which are resistant to conventional methods of therapy, as demonstrated by Soengas et al “Inactivation of the apoptosis effector Apaf-1 in malignant melanoma” Nature 409; 207-211(2001).
• experiments with Min mice have shown that inhibition of DNA methylation can suppress tumor initiation, Laird et. al. “Suppression of intestinal neoplasia by DNA hypomethylation” Cell 81; 197-205 (1995).
• DNA methylation analysis may provide novel means for cancer diagnosis.
• 5-methylcytosine is the most frequent covalent base modification in the DNA of eukaryotic cells. It plays a role, for example, in the regulation of the transcription, in genetic imprinting, and in tumorigenesis. Therefore, the identification of 5-methylcytosine as a component of genetic information is of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing since 5-methylcytosine has the same base pairing behavior as cytosine. Moreover, the epigenetic information carried by 5-methylcytosine is completely lost during PCR amplification.
• a relatively new and currently the most frequently used method for analyzing DNA for 5-methylcytosine is based upon the specific reaction of bisulfite with cytosine which, upon subsequent alkaline hydrolysis, is converted to uracil which corresponds to thymidine in its base pairing behavior.
• 5-methylcytosine remains unmodified under these conditions. Consequently, the original DNA is converted in such a manner that methylcytosine, which originally could not be distinguished from cytosine by its hybridization behavior, can now be detected as the only remaining cytosine using “normal” molecular biological techniques, for example, by amplification and hybridization or sequencing. All of these techniques are based on base pairing which can now be fully exploited.
• the prior art is defined by a method which encloses the DNA to be analyzed in an agarose matrix, thus preventing the diffusion and renaturation of the DNA (bisulfite only reacts with single-stranded DNA), and which replaces all precipitation and purification steps with fast dialysis (Olek A, Oswald J, Walter J. A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 1996 Dec. 15; 24(24):5064-6). Using this method, it is possible to analyze individual cells, which illustrates the potential of the method.
• Fluorescently labeled probes are often used for the scanning of immobilized DNA arrays.
• the simple attachment of Cy3 and Cy5 dyes to the 5′-OH of the specific probe are particularly suitable for fluorescence labels.
• the detection of the fluorescence of the hybridized probes may be carried out, for example via a confocal microscope. Cy3 and Cy5 dyes, besides many others, are commercially available.
• Matrix Assisted Laser Desorption Ionization Mass Spectrometry is a very efficient 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 thus transporting the analyte molecule into the vapor phase in an unfragmented manner.
• the analyte is ionized by collisions with matrix molecules.
• An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, the ions are accelerated at different rates. Smaller ions reach the detector sooner than bigger ones.
• MALDI-TOF spectrometry is excellently suited to 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 to nucleic acids is approximately 100 times worse than to peptides and decreases disproportionally with increasing fragment size.
• the ionization process via the matrix is considerably less efficient.
• the selection of the matrix plays an eminently important role.
• the object of the present invention is to provide the chemically modified DNA of genes associated with metastasis, as well as oligonucleotides and/or PNA-oligomers for detecting cytosine methylations, as well as a method which is particularly suitable for the diagnosis and/or therapy of genetic and epigenetic parameters of genes associated with metastasis.
• the present invention is based on the discovery that genetic and epigenetic parameters and, in particular, the cytosine methylation pattern of genes associated with metastasis are particularly suitable for the diagnosis and/or therapy of diseases associated with metastasis.
• nucleic acid containing a sequence of at least 18 bases in length of the chemically pretreated DNA of genes associated with metastasis according to one of Seq. ID No.1 through Seq. ID No. 198 and sequences complementary thereto and/or of genes according to one of the sequences of the genes according to table 1 and sequences complementary thereto.
• the respective data bank numbers accession numbers
• GenBank was used as the underlying data bank, which is located at the National Institute of Health, interact address www.ncbi.nlm.nih.gov.
• the object of the present invention is further achieved by an oligonucleotide or oligomer for detecting the cytosine methylation state in chemically pretreated DNA, containing at least one base sequence having a length of at least 13 nucleotides which hybridizes to a chemically pretreated DNA of genes associated with metastasis according to Seq. ID No. 1 through Seq. ID No. 198 and sequences complementary thereto and/or of genes according to one of the sequences of the genes according to table 1 and sequences complementary thereto.
• the oligomer probes according to the present invention constitute important and effective tools which, for the first time, make it possible to ascertain the genetic and epigenetic parameters of genes associated with metastasis.
• the base sequence of the oligomers preferably contains at least one CpG dinucleotide.
• the probes may also exist 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 present invention in which the cytosine of the CpG dinucleotide is the 5 th -9 th nucleotide from the 5′-end of the 13-mer; in the case of PNA-oligomers, it is preferred for the cytosine of the CpG dinucleotide to be the 4 th -6 th nucleotide from the 5′-end of the 9-mer.
• the oligomers according to the present invention are normally used in so called “sets” which contain at least one oligomer for each of the CpG dinucleotides of the sequences of Seq. ID No.1 through Seq. ID No. 198 and sequences complementary thereto and/or of genes according to one of the sequences of the genes according to table 1 and sequences complementary thereto.
• Preferred is a set which contains at least one oligomer for each of the CpG dinucleotides from one of Seq. ID No.1 through Seq. ID No. 198 and sequences complementary thereto and/or of genes according to one of the sequences of the genes according to table 1 and sequences complementary thereto.
• the present invention makes available a set of at least two oligonucleotides which can be used as so-called “primer oligonucleotides” for amplifying DNA sequences of one of Seq. ID No.1 through Seq. ID No. 198 and sequences complementary thereto and/or of genes according to one of the sequences of the genes according to table 1 and sequences complementary thereto, or segments thereof.
• At least one oligonucleotide is bound to a solid phase.
• the present invention moreover relates to a set of at least 10 n (oligonucleotides and/or PNA-oligomers) used for detecting the cytosine methylation state in chemically pretreated genomic DNA (Seq. ID No.1 through Seq. ID No. 198 and sequences complementary thereto and/or of genes according to one of the sequences of the genes according to table 1 and sequences complementry thereto).
• These probes enable diagnosis and/or therapy of genetic and epigenetic parameters of genes associated with metastasis.
• the set of oligomers may also be used for detecting single nucleotide polymorphisms (SNPs) in the chemically pretreated DNA of genes associated with metastasis according to one of Seq. ID No.1 through Seq. ID No. 198 and sequences complementary thereto of genes according to one of the sequences of the genes according to table 1 and sequences complementary thereto.
• SNPs single nucleotide polymorphisms
• a further subject matter of the present invention is a method for manufacturing an array fixed to a carrier material for analysis in connection with diseases associated with metastasis in which method at least one oligomer according to the present invention is coupled to a solid phase.
• Methods for manufacturing such arrays are known, for example, from U.S. Pat. No. 5,744,305 by means of solid-phase chemistry and photolabile protecting groups.
• kits which may be composed, for example, of a bisulfite-containing reagent, a set of primer oligonucleotides containing at least two oligonucleotides whose sequences in each case correspond or are complementary to an 18 base long segment of the base sequences specified in the appendix (Seq. ID No. 1 through Seq. ID No. 198 and sequences complementary thereto and/or of genes according to one of the sequences of the genes according to table I and sequences complementary thereto), oligonucleotides and/or PNA-oligomers as well as instructions for carrying out and evaluating the described method.
• a kit along the lines of the present invention can also contain only part of the aforementioned components.
• the genomic DNA to be analyzed is preferably obtained form usual sources of DNA such as cells or cell components, for example, cell lines, biopsies, blood, sputum, stool, urine, cerebral-spinal spinal fluid, tissue embedded in paraffm such as tissue from eyes, intestine, kidney, brain, heart, prostate, lung, breast or liver, histologic object slides, or combinations thereof.
• sources of DNA such as cells or cell components, for example, cell lines, biopsies, blood, sputum, stool, urine, cerebral-spinal spinal fluid, tissue embedded in paraffm such as tissue from eyes, intestine, kidney, brain, heart, prostate, lung, breast or liver, histologic object slides, or combinations thereof.
• the above described treatment of genomic DNA is preferably carried out with bisulfite (hydroben sulfite, disulfite) and subsequent alkaline hydrolysis which results in a conversion of non-methylated cytosine nucleobases to uracil or to another base which is dissimilar to cytosine in terms of base pairing behavior.
• Fragments of the chemically pretreated DNA are amplified, using sets of primer oligonucleotides according to the present invention, and a, preferably heat-stable polymerase. Because of statistical and practical considerations, preferably more than ten different fragments having a length of 100-2000 base pairs are amplified.
• the amplification of several DNA segments can be carried out simultaneously in one and the same reaction vessel. Usually, the amplification is carried out by means of a polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• At least one primer oligonucleotide is bonded to a solid phase during amplification.
• the different oligonucleotide and/or PNA oligomer sequences can be arranged on a plane solid phase in the form of a rectangular or hexagonal lattice, the solid phase surface preferably being composed of silicon, glass, polystyrene, aluminium, steel, iron, copper, nickel, silver, or gold, it being possible for other materials such as nitrocellulose or plastics to be used as well.
• the fragments obtained by means of the amplification can carry a directly or indirectly detectable label.
• the detection may be carried out and visualized by means of matrix assisted laser desorption/ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).
• MALDI matrix assisted laser desorption/ionization mass spectrometry
• ESI electron spray mass spectrometry
• Said oligonucleotides contain at least one base sequence having a length of 13 nucleotides which is reverse complementary or identical to a segment of the base sequences specified in the appendix, the segment containing at least one CpG dinucleotide.
• the cytosine of the CpG dinucleotide is the 5 th to 9 th nucleotide from the 5′-end of the 13-mer.
• One oligonucleotide exists for each CpG dinucleotide.
• the hybridized amplificates are detected.
• labels attached to the amplificates are identifiable at each position of the solid phase at which an oligonucleotide sequence is located.
• the labels of the amplificates are fluorescence labels, radionuclides, or detachable molecule fragments having a typical mass which can be detected in a mass spectrometer.
• the mass spectrometer is preferred for the detection of the amplificates, fragments of the amplificates or of probes which are complementary to the amplificates, it being possible for the detection to be carried out and visualized by means of matrix assisted laser desorption/ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).
• MALDI matrix assisted laser desorption/ionization mass spectrometry
• ESI electron spray mass spectrometry
• the produced fragments may have a single positive or negative net charge for better detectability in the mass spectrometer.
• the aforementioned method is preferably used for ascertaining genetic and/or epigenetic parameters of genes associated with metastasis.
• the oligomers according to the present invention or arrays thereof as well as a kit according to the present invention are intended to be used for the diagnosis and/or therapy of diseases associated with metastasis by analyzing methylation patterns of genes associated with metastasis.
• the method is preferably used for the diagnosis and/or therapy of important genetic and/or epigenctic parameters within genes associated with metastasis.
• the method according to the present invention is used, for example, for the diagnosis and/or therapy of solid tumours and cancer.
• nucleic acids according to the present invention of Seq. ID No. 1 through Seq. ID No. 198 and sequences complementary thereto and/or of genes according to one of the sequences of the genes according to table 1 and sequences complementary thereto can be used for the diagnosis and/or therapy of genetic and/or epigenetic parameters of genes associated with metastasis
• the present invention moreover relates to a method for manufacturing a diagnostic agent and/or therapeutic agent for the diagnosis and/or therapy of diseases associated with metastasis by analyzing methylation patterns of genes associated with metastasis, the diagnostic agent and/or therapeutic agent being characterized in that at least one nucleic acid according to the present invention is used for manufacturing it, possibly together with suitable additives and auxiliary agents.
• a further subject matter of the present invention relates to a diagnostic agent and/or therapeutic agent for diseases associated with metastasis by analyzing methylation patterns of genes associated with metastasis, the diagnostic agent and/or therapeutic agent containing at least one nucleic acid according to the present invention, possibly together with suitable additives and auxiliary agents.
• the present invention moreover relates to the diagnosis and/or prognosis of events which are disadvantageous to patients or individuals in which important genetic and/or epigenetic parameters within genes associated with metastasis said parameters obtained by means of the present invention may be compared to another set of genetic and/or epigenetic parameters, the differences serving as the basis for a diagnosis and/or prognosis of events which are disadvantageous to patients or individuals.
• hybridization is to be understood as a bond of an oligonucleotide to a completely complementary sequence along the lines of the WatsonCrick Crick base pairings in the sample DNA, forming a duplex structure.
• stringent hybridization conditions are those conditions in which a hybridization is carried out at 60° C. in 2.5 ⁇ SSC buffer, followed by several washing steps at 37° C. in a low buffer concentration, and remains stable.
• the term “functional variants” denotes all DNA sequences which are complementary to a DNA sequence, and which hybridize to the reference sequence under stringent conditions and have an activity similar to the corresponding polypeptide according to the present invention.
• “genetic parameters” are mutations and polymorphisms of genes associated with metastasis and sequences further required for their regulation.
• mutations are, in particular, insertions, deletions, point mutations, inversions and polymorphisms and, particularly preferred, SNPs (single nucleotide polymorphisms).
• epigenetic parameters are, in particular, cytosine methylations and further chemical modifications of DNA bases of genes associated with metastasis and sequences further required for their regulation.
• Further epigenetic parameters include, for example, the acetylation of histones which, however, cannot be directly analyzed using the described method but which, in turn, correlates with the DNA methylation.
• Sequences having odd sequence numbers exhibit in each case sequences of the chemically pretreated genomic DNAs of different genes associated with metastasis.
• Sequences having even sequence numbers exhibit in each case the sequences of the chemically pretreated genomic DNAs of genes associated with metastasis which are complementary to the preceeding sequences (e.g., the complementary sequence to Seq. ID No.1 is Seq. ID No.2, the complementary sequence to Seq. ID No.3 is Seq. ID No.4, etc.)
• Sequence ID Nos. 199 to 202 show the sequences of oligonucleotides used in Example 1.
• the following example relates to a fragment of a gene associated with metastasis, in this case, CD22 in which a specific CG-position is analyzed for its methylation status.
• the following example relates to a fragment of the gene CD22 in which a specific CG-position is to be analyzed for methylation.
• a genomic sequence is treated using bisulfite (hydrogen sulfite, disulfite) in such a manner that all cytosines which are not methylated at the 5-position of the base are modified in such a manner that a different base is substituted with regard to the base pairing behavior while the cytosines methylated at the 5-position remain unchanged.
• bisulfite hydrogen sulfite, disulfite
• the treated DNA sample is diluted with water or an aqueous solution.
• the DNA is subsequently desulfonated (10-30 min, 90-100 ° C.) at an alkaline pH value.
• the DNA sample is amplified in a polymerase chain reaction, preferably using a heatresistant DNA polymerase.
• cytosines of the gene CD22 are analyzed.
• a defined fragment having a length of 470 bp is amplified with the specific primer oligonucleotides TGTGTGTTGTTAAATGAAGA (Sequence ID No. 199) and ACACAAATATTAAAATTATC (Sequence ID No. 200).
• This amplificate serves as a sample which hybridizes to an oligonucleotide previously bonded to a solid phase, forming a duplex structure, for example TTGTTATACGTTTTGTTT (Sequence ID No.
• the cytosine to be detected being located at position 210 of the amplificate.
• the detection of the hybridization product is based on Cy3 and Cy5 fluorescently labelled primer oligonucleotides which have been used for the amplification.
• a hybridization reaction of the amplified DNA with the oligonucleotide takes place only if a methylated cytosine was present at this location in the bisulfite-treated DNA.
• the methylation status of the specific cytosine to be analyzed is inferred from the hybridization product.
• a sample of the amplificate is further hybridized to another oligonucleotide previously bonded to a solid phase.
• Said olignonucleotide is identical to the oligonucleotide previously used to analyze the methylation status of the sample, with the exception of the position in question.
• said oligonucleotide comprises a thymine base as opposed to a cytosine base i.e TTGTTATATGTTTTGTTT (Sequence ID No. 202). Therefore, the hybridisation reaction only takes place if an unmethylated cytosine was present at the position to be analysed.
• methylation patterns In order to relate the methylation patterns to one of the diseases associated with metastasis, it is initially required to analyze the DNA methylation patterns of a group of diseased and of a group of healthy patients. These analyses are carried out, for example, analogously to Example 1. The results obtained in this manner are stored in a database and the CpG dinucleotides which are methylated differently between the two groups are identified. This can be carried out by determining individual CpG methylation rates as can be done, for example, in a relatively imprecise manner, by sequencing or else, in a very precise manner, by a methylation-sensitive “primer extension reaction”. It is also possible for the entire methylation status to be analyzed simultaneously, and for the patterns to be compared, for example, by clustering analyses which can be carried out, for example, by a computer.
• Example 2 can be carried out, for example, for cancer and solid tumours.
• TABLE 1 List of preferred genes associated with metastasis according to the invention GenBank Entry No. Gene (http://www.ncbi.nim.nih.gov) CD20 L23418 FN1 M10905 SDC2 H04621 ACTG1 NM_001614 CDH2 NM_001792 CDH4 NM_001794 CDW52 NM_001803 ITGAX NM_000887 UTGB1 NM_002211 ITGB5 NM_002213 ITGB7 NM_000889 NEO1 NM_002499 PCDH1 NM_002587 ENPP1 NM_006208 RTN1 NM_021136 SELPLG NM_003006 TM4SF2 NM_004615 ITGAE NM_002208 SPTB NM_000347 ITGB1 NM_002211

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