CBX6 as epigenetic marker for the identification of immune cells, in particular memory B cells
The present invention relates to a method, in particular an in vitro method, for identifying memory B cells, comprising analyzing epigenetic modifications/properties of (including the methylation status) of at least one CpG position in the mammalian gene region for Chromobox protein homolog 6 (CBX6), wherein a demethylation or lack of methylation of said gene region is indicative for a memory B cell, when compared to a non-memory B cell. The analyses according to the invention can identify memory B cells on an epigenetic level and distinguish them from all other cells in complex samples, such as, for example, other blood or immune cells. The resent invention furthermore provides an improved method for quantifying memory B cells, in particular in complex samples. The method can be performed without a step of purifying and/or enriching cells, preferably in whole blood and/or non-trypsinized tissue.
1 . A method for producing an amplicon, the method comprising
a) bisulfite treating isolated genomic DNA from a mammalian cell sample to generate bisulfite treated DNA, and
b) producing the amplicon by amplifying from the bisulfite treated DNA of the mammalian gene region for chromobox protein homolog 6 (CBX6) comprising SEQ ID NO: 2, wherein at least one cytosine-phosphate-guanine (CpG) position in SEQ ID NO: 2 comprises bisulfite convertible cytosine, and
wherein the amplicon comprises TG at the respective CpG position comprising the bisulfite convertible cytosine.
2 . The method according to claim 1 , wherein said at least one CpG position is selected from the CpG at positions 91, 157, 171, 202, 240, 408, and 439 in SEQ ID NO: 2, and wherein the amplicon comprises TG at least one CpG position.
3 . The method of claim 1 , wherein the bisulfite convertible cytosine is detected by a method selected from a methylation specific enzymatic digest, bisulfite sequencing, promoter methylation analysis, CpG island methylation analysis, MSP, and other methods relying on a detection of amplified DNA.
4 . The method of claim 1 , wherein said sample is selected from a mammalian body fluid, human blood samples, a tissue, organ, cell type blood sample, a sample of blood lymphocytes or a fraction thereof.
5 . The method of claim 1 , wherein said method is performed without a step of purifying and/or enriching said cell sample.
6 . The method of claim 1 , wherein said cell sample is from a mammal that suffers from or is likely to suffer from autoimmune diseases, transplant rejections, infection diseases, cancer, and/or allergy.
7 . The method of claim 1 , wherein the method is performed using a kit comprising a) a bisulfite reagent, and b) materials for detecting the bisulfite convertible cytosine.
8 . The method according to claim 1 , wherein said gene region comprises a bisulfite convertible cytosine at CpG positions 157, 171, 202, 240, 408, and 439 in SEQ ID NO: 2, and wherein the amplicon comprises TG at the respective CpG positions 157, 171, 202, 240, 408, and 439 in SEQ ID NO: 2.
9 . The method according to claim 1 , wherein the mammalian cell sample is whole blood and/or non-trypsinized tissue.
10 . The method of claim 1 , wherein amplifying is performed with a polymerase chain reaction (PCR).
11 . The method of claim 10 , wherein the PCR is quantitative PCR (qPCR).
12 . The method of claim 10 , wherein the PCR is performed with one or more oligomers selected from SEQ ID NOs: 3-10.
13 . The method of claim 10 , wherein the PCR is performed with one or more oligomers selected from SEQ ID NOs: 5-7.
14 . The method of claim 10 , wherein the PCR is performed using a probe comprising the nucleic acid sequence of SEQ ID NO: 7.
15 . The method of claim 1 , further comprising c) detecting the amplicon with a probe comprising the nucleic acid sequence of SEQ ID NO: 7.