Epigenetic markers for the identification of blood sub-cells of type 1
Inventors
Olek, Sven • Turbachova, Ivana
Assignees
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Publication Number
US-12286675-B2
Publication Date
2025-04-29
Expiration Date
Abstract
The present invention relates to a method, in particular an in vitro method, for identifying CD3CD4 positive T lymphocytes of a mammal, wherein said method comprises analysing the methylation status of at least one CpG position in the CD3a/b/c/d/g genes, in particular their “upstream” regulatory regions, and in particular the promoter and other conserved regions of the gene cd3, wherein a demethylation of at least one CpG in the analyzed sample to at least 90% is indicative for memory and naive CD4 or/and memory and/or naive T lymphocytes. Furthermore, the present invention is directed at the use of DNA-methylation analysis of the genes CD3a/b/c/d for the detection and quality assurance and control of T lymphocytes. Furthermore, the present invention relates to a kit for performing the above methods as well as respective uses thereof. In a preferred embodiment, the present invention furthermore provides an improved method for analysing the methylation status of at least one CpG position in the gene CD3, allowing for a precise analysis even from sub-optimal quality samples, such as non-freshly obtained blood or serum samples.
Core Innovation
The invention provides a method for producing an amplicon from bisulfite treated human genomic DNA. Genomic DNA obtained from a human sample is bisulfite treated to produce bisulfite treated DNA, and a CD3 genetic region is amplified from the bisulfite treated DNA with at least one primer pair to produce the amplicon.
The amplicon comprises the sequence of SEQ ID NO: 43, SEQ ID NO: 55, or SEQ ID NO: 60. The amplified CD3 genetic region is directed to CD3D and/or CD3G upstream regulatory regions, including promoter and TLSDR (T lymphocyte specific demethylated regions) contexts, and DNA methylation status at CpG positions in these upstream regulatory regions is used to identify CD3+ T-lymphocyte subsets.
CD3 demethylation functions as a surrogate marker for CD3+ T cells and is used for detection and quality assurance/control from sub-optimal blood/serum samples. Additional CpG-methylation markers in specified genes are used together to enable unambiguous identification of all CD3+ T cells and to discriminate CD8 versus CD4 subsets using different marker sets.
Claims Coverage
The independent claim covers a bisulfite-to-amplicon workflow for specified CD3 genetic-region sequences, producing amplicons that include defined SEQ ID sequences. The claims further define amplification in CD3D/CD3G regulatory regions, methylation-status detection at CpG positions, selected human sample types, and required SEQ ID combinations within the amplicon.
Bisulfite treating genomic DNA and amplifying a CD3 genetic region to produce a specified amplicon
Bisulfite treating genomic DNA obtained from a human sample to produce bisulfite treated DNA, and amplifying a CD3 genetic region from the bisulfite treated DNA with at least one primer pair to produce an amplicon, wherein the amplicon comprises the sequence of SEQ ID NO: 43, SEQ ID NO: 55, or SEQ ID NO: 60.
Amplifying from upstream regulatory promoter regions of CD3D/CD3G
Amplifying the CD3 genetic region from a 5′ region upstream from the transcription start, and/or promoter region of the CD3D gene or CD3G gene.
Detecting methylation status of CpG positions
Detecting the methylation status of at least one CpG position using a selected methylation-detection technique.
Selecting the human sample type for genomic DNA
Selecting the sample as human body fluid, a human blood sample, tissue, an organ, a specific cell type blood sample, or blood lymphocytes.
Using the method for specified patient conditions
Providing the method for a human patient suffering from autoimmune diseases, transplant rejections, cancer, and/or allergy.
Requiring specific SEQ ID combinations within the amplicon
Performing the method such that the amplicon includes SEQ ID NO:55 together with one of SEQ ID NO:43 or SEQ ID NO:60.
Overall, the claims cover producing CD3-region amplicons from bisulfite treated human genomic DNA using defined primer-pair amplification targets that contain specified SEQ ID sequences, with further refinements that direct amplification to CD3D/CD3G promoter/upstream regulatory contexts, optionally detect CpG methylation status, constrain sample types, and limit the permitted SEQ ID composition of the amplicon.
Stated Advantages
Enables detection/quality assurance/control for identifying CD3+ T cells using CD3 demethylation as a surrogate marker.
Supports unambiguous identification of all CD3+ T cells using additional CpG-methylation markers.
Enables discrimination of CD8 versus CD4 using distinct marker sets.
Documented Applications
In vitro DNA-methylation assay to identify CD3+ T-lymphocyte subsets by analyzing demethylation at CpG positions in upstream regulatory regions of CD3D/CD3G/CD3E.
Detection and quality assurance/control from sub-optimal blood/serum samples.
Unambiguous identification of all CD3+ T cells using additional CpG-methylation markers in specified genes.
Discrimination of CD8 versus CD4 CD3+ T-cell subsets using specified marker sets.
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