MCC as epigenetic marker for the identification of immune cells, in particular basophil granulocytes
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Publication Number
US-11753683-B2
Publication Date
2023-09-12
Expiration Date
Abstract
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.
Core Innovation
The invention provides a method for producing an amplicon from a region of the mutated in colorectal cancer (MCC) gene. The method includes bisulfite treating isolated genomic DNA from a mammalian cell sample to generate bisulfite treated DNA, and producing the amplicon by amplifying from the bisulfite treated DNA. The amplifying is performed by polymerase chain reaction (PCR) using methylation-specific primers.
In the disclosed MCC region, the amplicon comprises CA at CA positions 186, 198, and 233 relative to SEQ ID NO:2. The disclosure further supports selecting additional CA positions within SEQ ID NO:2, including embodiments in which the amplicon comprises CA at one or more selected CA positions and embodiments requiring three or more CA positions from defined subsets.
The disclosure also provides epigenetic methylation/bisulfite convertibility differences at MCC gene CpG positions to identify and quantify basophil granulocytes in complex samples. It states that the MCC genomic region SEQ ID NO:1 (amplicon AMP2694) shows demethylation in basophils, while other immune cells are largely methylated, enabling discrimination.
Preferred sequence and assay embodiments are provided using specific bisulfite-converted target regions (SEQ ID NO:2 and SEQ ID NO:3) and primer/probe/amplicon sets identified by SEQ ID NOs:4–11. The disclosure further includes kit components and comparative evaluation information across purified cell types.
Claims Coverage
The partial content identifies one independent claim (clm-00001). The claim coverage focuses on a methylation-specific, bisulfite-based PCR workflow using a defined MCC gene region and requiring a specific CA pattern at defined CA positions relative to SEQ ID NO:2.
Bisulfite treatment of isolated genomic DNA from a mammalian cell sample
The method comprises bisulfite treating isolated genomic DNA from a mammalian cell sample to generate bisulfite treated DNA.
PCR amplification of a defined MCC gene region using methylation-specific primers
The method produces 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 polymerase chain reaction (PCR) using methylation-specific primers.
Amplicon CA pattern at CA positions 186, 198, and 233 relative to SEQ ID NO:2
The amplicon comprises CA at CA positions 186, 198, and 233 relative to SEQ ID NO:2.
Overall, the independent claim coverage is centered on producing a methylation-specific PCR amplicon from a defined mutated MCC gene region using bisulfite treated DNA, with an amplicon CA pattern at specified CA positions relative to SEQ ID NO:2. Dependent claims refine by specifying sample type, expanding or constraining required CA positions within SEQ ID NO:2, defining detection approaches, and identifying specific primer/oligomer sequence identifiers.
Stated Advantages
Enables discrimination based on epigenetic methylation/bisulfite convertibility differences at MCC gene CpG positions between basophils and other immune cells.
Documented Applications
Identification and quantification of basophil granulocytes in complex samples such as peripheral blood and tissues.
Diagnostic and monitoring applications related to immune status based on methylation-status detection using the MCC genomic region.
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