Method for the determination of the dna methylation level of a cpg position in identical cells within a tissue sample

Abstract

Aspects of the present invention relate to the determination of the DNA methylation level at one or more CpG position within cells of a defined type in a tissue sample. This methylation level is deduced from the total DNA methylation level of all cells of the sample and from the content of said cells of interest. In aspects of the invention, the cell content is determined by means of histopatholoy, staining methods, antibodies, expression analysis or DNA methylation analysis.

1 . Method for the determination of the DNA methylation level of one or more specific CpG positions of a subpopulation of cells within a tissue sample, comprising
 a) determining the cell content of said subpopulation of cells within the tissue sample in a quantitative or semi-quantitative manner,   b) measuring the total DNA methylation level of said one or more specific CpG positions within the tissue sample,   c) determining the DNA methylation level within the cells of said subpopulation of cells from the total DNA methylation level of said one or more specific CpG positions within the tissue sample and the cell content of said subpopulation of cells within the tissue sample.   
     
     
         2 . Method according to  claim 1 , characterized in that the cell content of said subpopulation of cells is determined by using staining methods or specific antibodies. 
     
     
         3 . Method according to  claim 1 , characterized in that the cell content of said subpopulation of cells is determined by histopathology. 
     
     
         4 . Method according to  claim 1 , characterized in that the cell content of said subpopulation of cells is determined by using expression analysis. 
     
     
         5 . Method according to  claim 1 , characterized in that the cell content of said subpopulation of cells is determined by DNA methylation analysis. 
     
     
         6 . Method according to  claim 5 , characterized in that the cell content of said subpopulation of cells is determined by
 a) measuring, in one or more loci, the total DNA amount present in the tissue sample,   b) measuring, in one or more loci, the amount of methylated DNA,   c) determining the cell content of said subpopulation of cells in the tissue sample, as the amount of methylated DNA within the total DNA.   
     
     
         7 . Method according to  claim 5 , characterized in that the cell content of said subpopulation of cells is determined by
 a) measuring, in one or more loci, the total DNA amount present in the tissue sample,   b) measuring, in one or more loci, the amount of unmethylated DNA, and   c) determining the cell content of said subpopulation of cells in the tissue sample, as the amount of unmethylated DNA within the total DNA.   
     
     
         8 . Method according to  claim 5 , characterized in that the cell content of said subpopulation of cells is determined by
 a) measuring, in one or more loci, the unmethylated DNA amount present in the tissue sample,   b) measuring, in one or more of said loci, the methylated DNA amount present in the tissue sample, and   c) determining the cell content of said subpopulation of cells of the tissue sample as the ratio of methylated DNA to unmethylated plus methylated DNA, or as the ratio of unmethylated DNA to unmethylated plus methylated DNA.   
     
     
         9 . Method according to  claim 5 , characterized in that the cell content of said subpopulation of cells is determined by
 a) measuring, in one or more loci, the ratio of methylated DNA to unmethylated DNA present in the tissue sample, or by measuring, in one or more loci, the ratio of unmethylated DNA to methylated DNA present in the tissue sample, and   b) determining the cell content of said subpopulation of cells of the tissue sample from the ratio of methylated DNA to unmethylated DNA, or from the ratio of unmethylated DNA to methylated DNA.   
     
     
         10 . Method according to  claim 5 , characterized in that the cell content of said subpopulation of cells is determined by
 a) measuring, in one or more loci, the amount of methylated DNA or the amount of unmethylated DNA present in the tissue sample,   b) determining the cell content of said subpopulation of cells of the tissue sample, from the amount of methylated DNA and the total volume or surface area of the tissue sample or from the amount of unmethylated DNA and the total volume or surface area of tissue sample.   
     
     
         11 . Method according to  claim 1 , characterized in that the cell content of said subpopulation of cells is determined by measuring the total DNA yield of a tissue sample in relation to the total volume or surface area of the tissue sample. 
     
     
         12 . Method according to  claim 1 , wherein the cells of said subpopulation of cells are disease-associated cells, preferably cells associated with a proliferative disease, in particular a cancer disease. 
     
     
         13 . Method according to  claim 1 , wherein DNA derived from the tissue sample is bisulfite treated for the measurement of the total DNA methylation level, the measurement of the amount of methylated DNA, and/or the measurement of the amount of unmethylated DNA. 
     
     
         14 . Method according to  claim 13 , characterized in that real-time PCR is performed for measurement subsequent to bisulfite treatment. 
     
     
         15 . Method according to  claim 14 , characterized in that the MethyLight™ method, the MethyLight™ ALGO™ method, or the QM™ assay is performed for measurement subsequent to bisulfite treatment. 
     
     
         16 . Method according to  claim 13 , characterized in that single nucleotide primer extension, mini-sequencing or sequencing is performed for measurement subsequent to bisulfite treatment. 
     
     
         17 . Method according to  claim 1 , characterized in that microarray hybridization is performed for measurement subsequent to bisulfite treatment. 
     
     
         18 . Method according to  claim 1 , characterized in that the total DNA methylation level, the amount of methylated DNA, and/or the amount of unmethylated is measured using methylation specific restriction enzymes, preferably the Mest evaluation method or the DMH method. 
     
     
         19 . Method for diagnosing a condition or disease, characterized by specific methylation levels of one or more methylation variable genomic DNA positions in a disease-associated cell of a tissue sample, comprising:
 a) obtaining a tissue sample comprising genomic DNA having one or more methylation variable positions in one or more regions thereof,   b) determining the disease-associated cell content within the tissue sample in a quantitative or semi-quantitative manner,   c) measuring the total DNA methylation level of one or more methylation variable genomic DNA positions of the tissue sample,   d) determining the DNA methylation level of one or more methylation variable genomic DNA positions within the disease-associated cells from the total DNA methylation level and the disease-associated cell content,   e) comparing said methylation level to that of corresponding reference tissue.   
     
     
         20 . Method for predicting treatment response or prognosis of disease for an individual, characterized by specific methylation levels of one or more methylation variable genomic DNA positions in a disease-associated cell of a tissue sample, comprising:
 a) obtaining a tissue sample comprising genomic DNA having one or more methylation variable positions in one or more regions thereof,   b) determining the disease-associated cell content within the tissue sample in a quantitative or semi-quantitative manner,   c) measuring the total DNA methylation level of one or more methylation variable genomic DNA positions of the tissue sample,   d) determining the DNA methylation level of one or more methylation variable genomic DNA positions within the disease-associated cells from the total DNA methylation level and the disease-associated cell content,   e) comparing said methylation level to that of corresponding reference tissue.   
     
     
         21 . Kit, comprising
 a) reagents for quantitative or semiquantitative determination of the cell content of a subpopulation of cells within a tissue sample,   b) reagents for measuring the total DNA methylation level of one or more specific CpG positions of said subpopulation of cells within the tissue sample, and   c) a container.   
     
     
         22 . Kit of  claim 21 , comprising operator instructions and/or algorithms to determine the methylation level within the tumor cells of a given sample. 
     
     
         23 . Kit of  claim 21  comprising one or more of the following
 a) one or more solution and/or one or more reagent for histological and/or immunological analysis, 
 b) one or more primer and/or one or more solution for DNA amplification, and 
 c) one or more primer, one or more oligonucleotide and/or one or more solution for detection of the DNA methylation level and/or the detection of the amount of methylated and/or unmethylated DNA. 
 
     
     
         24 . Kit, comprising one or more of the following:
 a container,   one or more primer suitable for the amplification of a subpopulation of cells specific DNA methylation marker to determine the cell content of said subpopulation of cells,   one or more primer suitable for the amplification of one or more fragments to determine the DNA methylation level of one or more specific CpG positions within the total DNA, and   operator instructions and/or algorithms to determine the methylation level within the tumor cells of a given sample.   
     
     
         25 . Kit according to  claim 21  for conducting a method according to one of the preceeding claims. 
     
     
         26 . Use of a method according to  claim 1  for analysis, characterization, classification, differentiation, grading, staging of a cell or tissue, diagnosis of proliferative disorders, or the predisposition to proliferative disorders, or combinations thereof. 
     
     
         27 . Use of a method according to  claim 1  for identifying an indication-specific target, wherein
 a) the DNA methylation level in disease-associated cells of a subpopulation of cells within a tissue sample is determined, 
 b) the DNA methylation level in corresponding healthy cells is determined; and 
 c) a indication-specific target is defined based on differences in the DNA methylation level of the DNA derived from the disease-associated cells in comparison to the DNA derived from the corresponding healthy cells. 
 
     
     
         28 . Use according to  claim 27 , wherein the indication-specific target is a protein, peptide or RNA. 
     
     
         29 . Use according to  claim 28 , wherein a per se known modulator of the protein, peptide or RNA is assigned to the specific indication of the diseased tissue. 
     
     
         30 . Use of the modulator assigned according to  claim 29  for preparing a pharmaceutical composition in case of a specific indication, or a specific cancer indication. 
     
     
         31 . Use of the method according to  claim 1  for diagnosis, prognosis or both of adverse events for patients or individuals, wherein the adverse events comprise at least one category selected from the group consisting of: undesired drug interactions; cancer diseases; CNS malfunctions; damage or disease; symptoms of aggression or behavioral disturbances; clinical; psychological and social consequences of brain damages; psychotic disturbances and personality disorders; dementia and/or associated syndromes; cardiovascular disease of the gastrointestinal tract; malfunction, damage or disease of the respiratory system; lesion, inflammation, infection, immunity and/or convalescence; malfunction, damage or disease of the body as an abnormality in the development process; malfunction, damage or disease of the skin, of the muscles, of the connective tissue or of the bones; endocrine and metabolic malfunction, damage or disease; and headaches or sexual malfunction. 
     
     
         32 . Use of the method according to  claim 1  for distinguishing subpopulations of cells or tissue or for investigating cell differentiation.