MVD as epigenetic marker for the identification of immune cells, in particular CD56+ NK cells
Inventors
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11788141-B2
Publication Date
2023-10-17
Expiration Date
Abstract
The present invention relates to a method, in particular an in vitro method, for identifying CD56+ NK cells, comprising analyzing the methylation status of at least one CpG position in the mammalian gene region for mevalonate (diphospho) decarboxylase (MVD), wherein a demethylation or lack of methylation of said gene region is indicative for a CD56+ NK cell, when compared to a non-CD56+ NK cell. The analyses according to the invention can identify CD56+ NK 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 present invention furthermore provides an improved method for quantifying CD56+ NK 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.
Core Innovation
The invention relates to an in vitro method of producing and detecting an amplicon from a bisulfite treated human mevalonate diphosphate decarboxylase (MVD) gene. Human genomic DNA from a sample comprising human immune cells is bisulfite treated to convert unmethylated cytosines to uracils, and an MVD gene region is amplified using methylation specific primers to produce an amplicon comprising thymine-guanine (TG) at TG positions 255 and 269 of SEQ ID NO: 2. The amplicon is detected using a probe comprising the nucleic acid sequence of SEQ ID NO: 8.
The method enables epigenetic identification and quantification by detecting bisulfite-convertibility/demethylation at at least one CpG motif within the human MVD gene region. CD56+ NK cells are characterized by near-complete demethylation of the relevant CpG motifs while other immune cell types are fully methylated, supporting discrimination of CD56+ NK cells in complex samples without purifying or enriching the immune cells.
The approach is described for whole blood and non-trypsinized tissue samples. Assay target and sequence-defined components include an MVD amplicon (AMP2674; SEQ ID NO: 1), target/bisulfite-converted sequences (SEQ ID NOs: 2–3), and qPCR primer/probe sets (SEQ ID NOs: 4–11), including a probe (SEQ ID NO: 8).
Claims Coverage
The document includes two independent claims covering 2 inventive features: bisulfite treatment and methylation-specific amplification of an MVD gene region, and probe-based detection of the resulting amplicon using SEQ ID NO: 8.
Bisulfite treated MVD genomic DNA conversion and methylation-specific amplification to TG positions
Bisulfite treating human genomic DNA from a sample comprising human immune cells to convert unmethylated cytosines to uracils; amplifying a region of the human MVD gene from the bisulfite treated DNA using methylation specific primers to produce an amplicon comprising thymine-guanine (TG) at TG positions 255 and 269 of SEQ ID NO: 2.
Probe-based detection of the MVD amplicon using SEQ ID NO: 8
Detecting the amplicon with a probe comprising the nucleic acid sequence of SEQ ID NO: 8.
Overall claim coverage centers on producing an MVD bisulfite-converted amplicon using methylation specific primers, with SEQ ID NO: 2 and TG positions 255 and 269, and detecting the resulting amplicon with a probe defined by SEQ ID NO: 8.
Stated Advantages
Enables discrimination of CD56+ NK cells in complex samples without purifying/enriching cells.
Supports epigenetic identification and quantification based on bisulfite convertibility/demethylation of CpG motifs within the human MVD gene region.
Provides cell-type specificity, with highest qPCR signal in CD56+ NK cells and lower/near-background signals for other immune populations.
Documented Applications
In vitro epigenetic identification and quantification of CD56+ NK cells using bisulfite convertibility/demethylation of CpG motifs within the human MVD gene region.
Detection in complex samples such as whole blood and non-trypsinized tissue without purifying/enriching immune cells.
Interested in licensing this patent?