MCC as epigenetic marker for the identification of immune cells, in particular basophil granulocytes
The present invention relates to a method, in particular an in vitro method, for identifying basophil granulocytes, comprising analyzing a modification such as for example the methylation status of at least one CpG position in the mammalian gene region for the gene “mutated in colorectal cancer” (MCC), wherein a demethylation or lack of modification or methylation of said gene region is indicative for a basophil granulocyte, when compared to a non-basophil granulocyte, or any other cell type in the peripheral blood or in other tissues. The analyses according to the invention can identify basophil granulocytes on an epigenetic level and distinguish them from all other cells in complex samples, such as, for example, other blood or immune cells. The present invention furthermore provides an improved method for quantifying basophil granulocytes, in particular in complex samples. The method can be performed with or without a step of purifying and/or enriching cells, preferably in whole blood and/or non-trypsinized tissue. Finally, the invention provides primers, probes and amplicons based on bisulfite treated nucleic acids.
1. A method for producing an amplicon from a region of mutated in colorectal cancer (MCC) gene, 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 the region of the MCC gene comprising nucleotides 57 to 234 of SEQ ID NO: 1 prior to bisulfite treatment, wherein the amplifying is performed with a polymerase chain reaction (PCR) using methylation-specific primers, and
wherein the amplicon comprises CA at CA positions 186, 198, and 233 relative to SEQ ID NO: 2.
2. The method according to claim 1 , wherein the amplicon further comprises CA at one or more CA positions selected from positions 57, 69, 76, 80, 119, 136, 142, 162, 339, 351, and 362 relative to SEQ ID NO: 2.
3. The method according to claim 1 , further comprising detecting the CA by a method selected from a methylation specific enzymatic digest, bisulfite sequencing, promoter methylation analysis, CpG island methylation analysis, MSP, HeavyMethyl, MethyLight, and Ms-SnuPE.
4. The method according to claim 1 , wherein said cell sample is obtained from a body fluid, a tissue, or an organ.
5. The method according to claim 1 , wherein said method is performed without a step of purifying and/or enriching said cell sample.
6. The method according to 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 CA.
8. The method of claim 1 , wherein the amplifying uses an oligomer comprising the sequence of any one of SEQ ID NOs: 4-7 or 11, or wherein the method produces the amplicon comprising SEQ ID NO: 2.
9. The method according to claim 1 , wherein the amplicon further comprises CA at two or more CA positions selected from CA positions 57, 69, 76, 80, 119, 136, 142, 162, 339, 351, and 362 of SEQ ID NO: 2.
10. The method according to claim 1 , wherein the mammalian cell sample is whole blood and/or non-trypsinized tissue.
11. The method of claim 1 , wherein the amplicon further comprises CA at three or more CA positions selected from CA positions 57, 69, 76, 80, 119, 136, 142, and 162 of SEQ ID NO: 2.
12. The method of claim 1 , wherein the amplicon further comprises CA at CA positions 339, 351, and 362 of SEQ ID NO: 2.
13. The method of claim 1 , wherein the amplifying uses an oligomer of SEQ ID NO: 11.
14. The method of claim 1 , wherein the amplicon comprises SEQ ID NO: 2.
15. The method of claim 1 , wherein the region of the MCC gene comprises nucleotides 1 to 234 of SEQ ID NO: 1 prior to bisulfite treatment.