PARK2 as epigenetic marker for the identification of immune cells, in particular monocytes

Inventors

Olek, Sven

Assignees

Precision for Medicine GmbH

Abstract

The present invention relates to a method, in particular an in vitro method, for identifying monocytes, comprising analyzing the methylation status of at least one CpG position in the mammalian gene region for parkin RBR E3 ubiquitin protein ligase (PARK2), wherein a de-methylation or lack of methylation of said gene region is indicative for a monocyte, when compared to a non-monocyte cell. The analyses according to the invention can identify monocytes 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 monocytes, 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. Also claimed are kits and specific oligonucleotides for use as primers or probes.

Core Innovation

The invention relates to epigenetic identification and quantification of human monocytes using bisulfite convertibility at the parkin RBR E3 ubiquitin protein ligase (PARK2) genomic region. Unmethylated cytosines are converted to uracils after bisulfite treating, so that demethylation is represented by a cytosine-adenine (CA) signature at defined positions within a PARK2 target region. The method focuses on detecting an amplicon produced from a PARK2 genomic region and identifying the presence of CA at specified CA positions within a bisulfite-converted reference amplicon.

A specific PARK2 genomic amplicon is targeted, including an amplicon reference comprising SEQ ID NO: 2 or SEQ ID NO: 3, and detection uses a probe comprising the nucleic acid sequence of SEQ ID NO: 8. The invention defines particular CA position signatures, including CA at CA positions 172, 178, and 182 and, in other embodiments, CA at additional CA positions. In some embodiments, the qPCR amplicon comprises nucleotides 165-190 of SEQ ID NO: 2 and has CA at specified CA positions relative to SEQ ID NO: 2.

The disclosed approach supports monocyte cell-type specificity by reporting that demethylation patterns at the selected PARK2 CpG positions are strongly monocyte-specific versus other immune cells. Quantification is described in terms of detecting PARK2 amplicon signals and using ratios to a control gene such as GAPDH and copy-based qPCR measurements to distinguish monocytes from CD14+ monocytes, classical and non-classical, and other cell types. The method further includes embodiments that detect methylation status of at least one CpG position and, in some aspects, methylation status of CpG positions derived from the amplicon.

Claims Coverage

The independent claims cover producing and detecting bisulfite-treated PARK2 genomic-region amplicons with qPCR and probe-based detection using SEQ ID NO: 8, with three independent sets of inventive requirements covering defined CA signatures and defined target regions or amplified sequences, and a monocyte-sample-specific version. Across the independent claims, the inventive features are organized around bisulfite treating of immune-cell DNA, qPCR amplification of defined PARK2 regions, and detecting with a probe comprising SEQ ID NO: 8, together with CA position signature constraints.

Collectively, the independent claims require producing and detecting bisulfite-converted PARK2 genomic-region amplicons from human immune cells or specifically from human monocyte samples, using qPCR amplification and probe-based detection with a probe comprising the nucleic acid sequence of SEQ ID NO: 8. The claim scope is further defined by CA position signature constraints on SEQ ID NO: 2 and by embodiments that constrain the amplicon to specific SEQ ID NO: 2 nucleotide ranges, thereby linking the measured CA pattern to the PARK2 epigenetic marker used for monocyte identification.

Stated Advantages

Documented Applications