Epigenetic marker for the identification of T lymphocytes
The invention relates to a method for identifying a specific type and/or state of a mammalian cell in a sample, the method comprising a) analyzing the relative amount of accessible chromatin in regions that are specific for a cell-type and/or cellular state in the genome of the cell, b) comparing the relative amount of accessible chromatin in the regions with the relative amount of accessible chromatin in regions in the genome of the cell that are unspecific for the cell-type and/or cellular state, and c) deducing the specific type and/or state of said mammalian cell in the sample based on such comparison. The identifying further comprises a relative quantification of the specific cell type and/or state. The method can further comprise a diagnosis of a predisposition to a disease or a disease based on such identification. Kits and markers in regions of accessible chromatin are also described.
1. A method for identifying a T lymphocyte in a sample of cells obtained from a mammal, the method comprising:
a) quantifying an amount of accessible chromatin in regions of a CD3 gene in the genome of said cells by bisulfite treatment and quantitative polymerase chain reaction (qPCR);
b) quantifying an amount of accessible chromatin in regions of a housekeeping gene by bisulfite treatment and qPCR;
c) carrying out a first step of normalizing said amount of accessible chromatin in said CD3 gene and said housekeeping gene to a known number of copies of a plasmid containing sequences equivalent to bisulfite-converted methylated versions of said CD3 regions and said housekeeping regions;
d) carrying out a second step of normalizing said amount of accessible chromatin in said CD3 gene and said housekeeping gene to a known number of copies of a plasmid containing sequences equivalent to bisulfite-converted non-methylated versions of said CD3 regions and said housekeeping regions; and
e) concluding the presence or absence of a T lymphocyte in said sample based on said first and second steps of normalizing.
2. The method according to claim 1 , wherein said concluding further comprises a quantification of the cells based on said amounts of accessible chromatin in said regions as determined.
3. The method according to claim 1 , wherein said housekeeping gene is selected from a gene encoding actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), aldolase, hexokinase, and cyclophilin.
4. The method, according to claim 3 , wherein said housekeeping gene is GAPDH.
5. The method according to claim 1 , wherein the sample is selected from the group consisting of blood or fractions thereof, saliva, buccal, tears, semen, urine, sweat, faecal material, skin and hair.