Patent Application
20070026393
The invention concerns a set of oligonucleotides as probes for the detection of relevant variations of DNA methylation in a target group of genes, the use thereof for the detection of gene variants with respect to DNA methylation, a medical device which uses a set of oligonucleotides, a method for the investigation of the methylation state of an individual as well as a method for the construction of a model for evaluating the probability of occurrence of a health problem in an individual.
The levels of observation that have been well studied in molecular biology according to developments in methods in recent years include the genes themselves, the transcription of these genes into RNA and the translation to proteins therefrom. During the course of development of an individual, which gene is turned on and how the activation and inhibition of certain genes in certain cells and tissues are controlled can be correlated with the extent and nature of the methylation of the genes or of the genome. In this regard, pathogenic states are also correlated with a modified methylation pattern of individual genes or of the genome. The present invention describes sets of oligomers and a method for the detection of relevant variations of the DNA methylation in a target group of genes, which are associated with adverse events for patients/individuals or are associated with specific disorders.
5-Methylcytosine is the most frequent 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 thus of considerable interest. 5-Methylcytosine positions, however, cannot be identified by sequencing, since 5-methylcytosine has the same base-pairing behavior as cytosine. In addition, in the case of a PCR amplification, the epigenetic information which is borne by the 5-methylcytosines is completely lost.
A relatively new method that in the meantime has become the most widely used me-method for investigating DNA for 5-methylcytosine is based on the specific reaction of bisulfite with cytosine, which, after subsequent alkaline hydrolysis, is then converted to uracil, which corresponds in its base-pairing behavior to thymidine. In contrast, 5-methylcytosine is not modified under these conditions. Thus, the original DNA is converted so that methylcytosine, which originally cannot be distinguished from cytosine by its hybridization behavior, can now be detected by “standard” molecular biology techniques as the only remaining cytosine, for example, by amplification and hybridization or sequencing. The prior art, which concerns sensitivity, is defined by a method that incorporates the DNA to be investigated in an agarose matrix, so that the diffusion and renaturation of the DNA is prevented (bisulfite reacts only on single-stranded DNA) and all precipitation and purification steps are replaced by rapid dialysis (Olek, A. et al., Nucl. Acids Res. 1996, 24, 5064-5066). Individual cells can be investigated by this method, which illustrates the potential of the method. Of course, up until now, only individual regions of up to approximately 3000 base pairs long have been investigated; a global investigation of cells for thousands of possible methylation analyses is not possible. Of course, this method also cannot reliably analyze very small fragments of small quantities of sample. These are lost despite the protection from diffusion through the matrix.
An overview of other known possibilities for detecting 5-methylcytosines can be derived from the following review article: Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998, 26, 2255.
With just a few exceptions (e.g. Zechnigk, M. et al., Eur. J. Hum. Gen. 1997, 5, 94-98), the bisulfite technique has only been applied in research up to now. However, short, specific segments of a known gene are always amplified after a bisulfite treatment and either completely sequenced (Olek, A. und Walter, J., Nat. Genet. 1997, 17, 275-276) or individual cytosine positions are detected by a “primer extension reaktion” (Gonzalgo, M. L. and Jones, P. A., Nucl. Acids Res. 1997, 25, 2529-2531, WO Patent 95-00669) or an enzyme step (Xiong, Z. and Laird, P. W., Nucl. Acids. Res. 1997, 25, 2532-2534). Detection by hybridization has also been described (Olek et al., WO 99 28498).
Other publications which are concerned with the application of the bisulfite technique for the detection of methylation in the case of individual genes are: Xiong, Z. and Laird, P. W. (1997), Nucl. Acids Res. 25, 2532; Gonzalgo, M. L. and Jones, P. A. (1997), Nucl. Acids Res. 25, 2529; Grigg, S. and Clark, S. (1994), Bioassays 16, 431; Zeschnik, M. et al. (1997), Human Molecular Genetics 6, 387; Teil, R. et al. (1994), Nucl. Acids Res. 22, 695; Martin, V. et al. (1995), Gene 157, 261; WO 97 46705, WO 95 15373 and WO 45560.
An overview of the state of the art in oligomer array production can be derived also from a special issue of Nature Genetics which appeared in January 1999 (Nature Genetics Supplement, Volume 21, January 1999), the literature cited therein and U.S. Pat. No. 5,994,065 on methods for the production of solid supports for target molecules such as oligonucleotides in the case of reduced nonspecific background signal.
Probes with multiple fluorescent labels are used for scanning an immobilized DNA array. The fluorescence of the hybridized probes is detected, for example, by means of confocal optics.
More recent methods for the detection of mutations, which also can be used, in principle but with several modifications, for methylation analyses after the bisulfite treatment of DNA samples, are listed below:
Genomic DNA is obtained from DNA of cells, tissue or other test samples by standard methods. This standard methodology is found in references such as Fritsch and Maniatis, eds., Molecular Cloning: A Laboratory Manual, 1989.
At present, it is not state of the art to investigate large quantities of samples relative to a multiple number of methylation positions that are important for diseases.
The object of the present invention is to make up sets of oligomer probes that bind to genes which are of importance relative to adverse events for patients or relative to specific groups of diseases, so that comprehensive prognostic information will be possible for the patient in question by analysis of the respective methylation state. The data detected in this way are combined to make up methylation patterns. In addition, a method will be created which makes possible to a great extent the analysis of methylation positions with the use of the above-mentioned oligomer probes.
The object is [solved] according to the invention by a set of nucleotide probes and a method for the investigation of the methylation profile of a patient or individual. The presence or absence of the relevant methylation variants from the target group of genes is detected by means of the set of oligonucleotide probes and/or by means of the method. A diagnosis is made from the methylation pattern by comparing it to methylation patterns in a database, which have already been assigned to specific phenotypes, prognoses or effects on the patient.
This object is solved according to the invention by a set of nucleotide probes according to one or more of the claims. In addition, to solve the object, a method or a device can be utilized, which contains a set of the above-mentioned nucleotide probes. Advantageous embodiments of the invention are characterized in the respective subclaims.
The set of oligonucleotide probes according to the invention or a device which contains this set preferably serves for the prognosis and planned treatment of patients who suffer from the consequences of subsequent adverse events or in whom there is a risk of the occurrence of the following adverse events:
This list is not conclusive and it is clear to the person of average skill in the art that the concept of the invention is applicable to any disease or malfunction of an organism or individual.
The oligomer probes comprise sequences which bind to genes that are associated with these adverse events. The oligomer probes bind to sequences as they are present after a treatment of the DNA sample which converts unmethylated cytosine to uracil. The genes are listed in detail in the claims. According to the invention, these genes belong to at least one of the following protein functions: enzyme, transport, storage, structure, immunity, neuronal transmission, growth and differentiation.
The method will be described in the following for an evaluation of whether adverse events will occur or will probably occur in a patient, an individual or a population, by using the set of oligomer probes. In the first step, a DNA sample is taken from the patient or individual, in whom an adverse event was diagnosed not and from a control group in whom the event was diagnosed. In the second step of the method, the DNA samples, which have been pretreated by means of the solution of a bisulfite, hydrogen sulfite or disulfite, are hybridized with a set of oligonucleotides as probes, which bind to the sequences in which a cytosine methylation is potentially present after bisulfite treatment within the respective target group of genes, in order to detect relevant gene variants with respect to DNA methylation. A hybridization pattern results, which is translated into a methylation pattern of the genes of the respective DNA sample in the third step of the method.
The procedure would be the same if one wanted to construct a model for evaluating risks for patients or patient groups.
According to the invention, these genes belong to at least one of the following protein functions: enzyme, transport, storage, structure, immunity, neuronal transmission, growth and differentiation. Said genes are associated with a multiple number of adverse events, including:
The oligonucleotide probes are characterized by the fact that they are complementary to the DNA sequences of the target group of genes or correspond to them as they are present after a chemical treatment which converts unmethylated cytosines to uracil, preferably a treatment with sodium bisulfite.
In the last step of the method, the frequency of alleles with different methylation patterns is calculated and compared with the frequencies of alleles in patients and individuals with adverse events and the corresponding control group.
Preferably, at least one of the steps of the method will be conducted with a computer.
In a preferred variant of the method, the methylation pattern of an individual is compared with the entries already present in the database or with a model derived therefrom, in order to evaluate the risk of the occurrence of adverse events.
The set of oligonucleotides is preferably comprised of oligonucleotides, which are arranged on a support at known loci in a rectangular or hexagonal grid. This support is comprised of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver, or gold.
A technical medical device which contains said set of oligonucleotides is preferably used for the detection of gene variants relative to methylation and different gene expression.
In a preferred variant of the method, said set of oligonucleotides or a device that contains them is used for predicting probable therapeutic consequences or adverse events as a consequence of a therapeutic intervention or as a consequence of taking specific medications.
In a preferred variant of the method, said set of oligonucleotides or a device is used for predicting probable symptoms when the above-listed adverse events occur and for predicting the probability of the occurrence of consequential disorders or other symptoms.
In another preferred variant of the method, said set of oligonucleotides or a device is used for the following objectives:
Another subject of the present invention is a kit for conducting an assay, with which the risk of a patient or individual of being subject to adverse events can be estimated. This kit comprises possibilities for testing for the presence or absence of relevant variations with respect to DNA methylation of the above-listed genes in a sample of genomic DNA. Reagents for use in the detection method are also included, such as well script, which describes the probability of a patient or individual of experiencing to undesired events.
The object of the invention is thus solved by a set of oligonucleotides as probes for the detection of relevant variations of DNA methylation in a target group of genes, characterized in that the oligonucleotides are complementary to the DNA sequence of the target group of genes or correspond to them as they are present after a chemical treatment which converts unmethylated cytosines to uracil, wherein the target group essentially comprises genes that are associated with a health problem of an individual.
It is preferred according to the invention that the set of oligonucleotides is characterized in that the health problems are: undesired drug interactions; cancer; CNS malfunctions, damage or disease; symptoms of aggression or behavioral disturbances; clinical, psychological and social consequences of a brain lesion; dementia and/or associated syndromes; psychotic disturbances and personality disorders; cardiovascular disease, malfunction or damage; malfunction, damage or disease of the gastrointestinal tract; damage or disease of the respiratory system; lesion, inflammation, infection, immunity and/or convalescence; malfunction, damage or disease of the body as a consequence of an abnormality in the development process; malfunction, damage or disorder of the skin, the muscles, the connective tissue or the bones; endocrine and metabolic malfunction; headaches; sexual malfunctions.
It is additionally preferred that the set of oligonucleotides is characterized in that the genes that are associated with undesired drug interactions are selected from Table 1, the genes that are associated with cancer are selected from Table 2, the genes associated with symptoms and consequences of CNS malfunction are selected from Table 3, the genes associated with symptoms of aggression or behavioral disturbances are selected from Table 4, the genes associated with the consequences of clinical, psychological and social consequences of a brain lesion are selected from Table 5, the syndromes associated with dementia and/or associated syndromes are selected from Table 6, the genes that are associated with psychotic disturbances and personality disorders are selected from Table 7, the genes associated with cardiovascular disease, malfunction or damage are selected from Table 8, the genes associated with malfunction, damage or disease of the gastrointestinal tract are selected from Table 9, the genes associated with malfunction, damage or disease of the respiratory system are selected from Table 10, the genes associated with lesion, inflammation, infection, immunity and/or convalescence are selected from Table 11, the genes associated with malfunction, damage or disease of the body as a consequence of an abnormality in the development process are selected from Table 12, the genes associated with a malfunction, damage or disease of the skin, the muscles, the connective tissue or the bones are selected from Table 13, the genes associated with endocrine and metabolic malfunction, damage or disease are selected from Table 14, the genes that are associated with headaches are selected from Table 15, and the genes that are associated with sexual malfunctions are selected from Table 16.
According to the invention, a set of oligonucleotides is preferred, in which oligonucleotides with up to 5% of the listed genes are not included.
In particular, a set of oligonucleotides is preferred, in which oligonucleotides with at least 95% of the listed genes are included, together with a limited number of additional, unlisted oligonucleotides.
A set of oligonucleotides is advantageous, in which up to 5% of the corresponding oligonucleotides of the listed genes are replaced by a complete set of 25% of other unlisted oligonucleotides.
In addition, a set of oligonucleotides for a target group of genes is advantageous, in which the chemically pretreated DNA sequence of the genes to be detected coincides at least up to 95% with the correspondingly pretreated DNA sequence of the genes from the above list.
A set of oligonucleotides is particularly advantageous, wherein the chemical pre-treatment is conducted by means of the solution of a bisulfite, hydrogen sulfite or disulfite.
A set of oligonucleotides that consists of a subgroup of the target group of genes is particularly advantageous.
According to the invention, a set of oligonucleotides is preferred in which the oligonucleotides are arranged on a support at known loci in a rectangular or hexagonal grid.
In addition, a set of oligonucleotides is preferred, wherein the oligonucleotides are arranged on a support, which is comprised of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold.
A set is particularly preferred, wherein the oligonucleotide probes are labeled via their mass, electrostatics, charge or fluorescence, or are labeled with radionuclides.
In particular, a set of oligonucleotides is preferably used in a biological investigation for the detection of said gene variants relative to DNA methylation.
A technical medical device is advantageous, which contains a set of oligonucleotides according to the invention, for use in an investigation for the detection of said gene variants, particularly as an indication of a higher risk of a patient or individual of developing symptoms and consequential signs of cancer or as an indication for a higher risk of the development of CNS malfunction, damage or disorder or for the patient or individual to experience symptoms and consequences of CNS malfunction, damage or disorder.
A technical medical device is particularly advantageous, which contains a set of oligonucleotides according to the invention, for use in an investigation for the detection of varying gene expression and/or for the prognosis and for the management of patients who suffer from the risk of developing symptoms and consequential signs of cancer and/or for use in an investigation of whether a patient or individual may have developed CNS malfunction, damage or disorder or whether it is probable that the patient or individual will experience the symptoms and consequences of CNS malfunction, damage or disorder.
According to the invention, a method for the investigation of the DNA methylation profile of a patient or an individual, which detects the presence or lack of the relevant methylation variants from the target group of genes is preferred in which a chemically pretreated nucleic acid sample of said patient or individual is hybridized to a set of oligonucleotides according to the invention and relating the hybridization pattern with the variations.
Particularly preferred is the use of a set of oligonucleotides according to the invention or a device according to the invention for the prognosis and/or planned treatment in patients, who suffer from a health problem or in whom the risk of onset of a health problem exists.
The use according to the invention is particularly advantageous for the prognosis and/or planned treatment for patients who suffer from the consequences of undesired drug interactions or in whom the risk of the occurrence of undesired drug interactions exists, who suffer from the risk of developing symptoms and consequential signs of cancer, who are affected by an increased risk of CNS malfunction, damage or disorder, who suffer from the consequences of symptoms of aggression or behavioral disturbances or in whom exists the risk of the occurrence of symptoms of aggression or behavioral disturbances, who suffer from the consequences of clinical, psychological and social consequences of a brain lesion or in whom exists the risk of the occurrence of consequences of clinical, psychological and social consequences of a brain lesion, who suffer from dementia and/or associated syndromes or in whom exists the risk of the occurrence of dementia and/or associated syndromes, who suffer from symptoms and consequences of psychotic disorders and personality disturbances or in whom the risk exists of the occurrence of psychotic disorders and personality disturbances, who suffer from symptoms or consequences of cardiovascular disease, malfunction or damage or in whom the risk exists of the occurrence of symptoms or consequences of cardiovascular disease, malfunction or damage, who suffer from symptoms and consequences of the malfunction, damage or disease of the gastrointestinal tract or in whom the risk exists of the occurrence of symptoms and consequences of the malfunction, damage or disease of the gastrointestinal tract, who experience clinical or social consequences that result from the malfunction, damage or disease of the respiratory system or in whom the risk exists of clinical or social consequences that result from malfunction, damage or disease of the respiratory system, who suffer from the symptoms and consequences of lesion, inflammation, infection, immunity and/or convalescence or in whom the risk exists of the occurrence of symptoms and consequences of lesion, inflammation, infection, immunity and/or convalescence, who suffer from the consequences of malfunction, damage or disorder of the body as a consequence of an abnormality in the development process or in whom the risk exists of the occurrence of malfunction, damage or disorder of the body as a consequence of an abnormality in the development process, who suffer from the consequences of a malfunction, damage or disorder of the skin, the muscles, the connective tissue or the bones or in whom the risk exists of the occurrence of a malfunction, damage or disorder of the skin, the muscles, the connective tissue or the bones, who suffer from the consequences of endocrine and metabolic malfunction, damage or disorder or in whom the risk exists of the occurrence of endocrine and metabolic malfunction, damage or disorder, who suffer from the consequences of headaches or in whom the risk exists of the occurrence of headaches, and/or who suffer from the consequences of sexual malfunctions or in whom the risk exists of the occurrence of sexual malfunctions.
The use of a set of oligonucleotides according to the invention or of a device according to the invention is preferred for predicting the probable therapeutic consequences of undesired drug interactions as a consequence of a therapeutic intervention and/or for predicting the probable therapeutic consequences and adverse results as a consequence of a therapeutic intervention.
In addition, the use of a set of oligonucleotides according to the invention or a device according to the invention is preferred for predicting probable therapeutic risks or of undesired drug interactions as a consequence of taking specific medications.
The use of a set of oligonucleotides according to the invention or of a device according to the invention is particularly preferred for predicting the probable symptoms if undesired drug interactions occur and the probability of the occurrence of consequential disorders or other symptoms and/or for predicting probable patterns of disease symptoms and the probability of the occurrence of consequential disorders or other symptoms.
The use of a set of oligonucleotides according to the invention or a device according to the invention is advantageous for the development of new strategies in therapeutic intervention and in clinical studies and/or for the prognosis or management of patients, who suffer from developing symptoms of aggression or behavioral disturbances or who belong to risk groups for said disturbances, and/or for modeling and evaluating the effects of disorders and providing precautionary measures health measures for individuals, ethnic groups, patient groups, and populations, and/or for generating a model in order to estimate the risk for individuals, ethnic groups, patient groups, and populations, for developing symptoms and consequential signs of cancer, and/or for generating a model for evaluating the risk of the development of symptoms and consequential signs of CNS malfunction, damage or disease, and/or for optimizing the therapeutic intervention.
A method is particularly advantageous for creating a model for evaluating whether a health problem will occur or will probably occur in a patient, an individual, or ethnic groups, which comprises the following steps:
A method according to the invention is preferred, wherein the health problem is selected from: undesired drug interactions; cancer; symptoms and consequential signs of CNS malfunction, damage or disorder; developing symptoms of aggression or behavioral disturbances; consequences of clinical, psychological and social consequences of a brain lesion; dementia and/or associated syndromes; psychotic disturbances and personality disorders; symptoms or consequences of cardiovascular disorder, malfunction or damage; symptoms and consequences of malfunction, damage or disease of the gastrointestinal tract; malfunction, damage or disease of the respiratory system; symptoms and consequences of lesion, inflammation, infection, immunity and/or convalescence, malfunction, damage or disease of the body as a consequence of an abnormality in the development process; malfunction, damage or disorder of the skin, the muscles, the connective tissue or the bones; endocrine and metabolic malfunction, damage or disorder; headaches; sexual malfunctions.
A method is of advantage for evaluating whether a specific individual bears the risk of the occurrence of a health problem, wherein the methylation profile is compared with the model constructed according to the invention.
A method is particularly of advantage according to which at least one of the steps is conducted by a computer.
In addition, a configured assay [hit] is advantageous for utilizing the estimation of the risk of a patient or individual of experiencing adverse events and/or health problems, said kit containing:
A configured assay [kit] is particularly advantageous for utilizing the estimation of the risk of a patient or an individual of experiencing adverse events and/or health problems, characterized in that the adverse event or the health problem is selected from the following:
symptoms and consequences of lesion, inflammation, infection, immunity and/or convalescence; malfunction, damage or disease of the body as a consequence of an abnormality in the development process; malfunction, damage or disorder of the skin, the muscles, the connective tissue or the bones; endocrine and metabolic malfunction, damage or disorder; headaches; sexual malfunctions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 100190588 | Apr 2000 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/DE01/01486 | 4/6/2001 | WO | 7/11/2003 |
