Method for epigenetic immune cell detection and counting in human blood samples
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Publication Number
US-12006546-B2
Publication Date
2024-06-11
Expiration Date
Abstract
The present invention relates to improved methods for epigenetic blood and immune cell detection and counting, and respective uses and kits.
Core Innovation
The invention relates to an epigenetic qPCR method for producing and detecting a panel of amplicons from human blood immune-cell samples. Unmethylated cytosines in isolated genomic DNA are converted by bisulfite treating, and the bisulfite treated DNA is amplified and/or quantified by qPCR to generate a panel of amplicons. The method uses a thymidine-cytosine (TpG) template to carry targets of converted genomic regions corresponding to CD3gamma/delta, CD4, CD8beta, lipoprotein receptor-related protein 5 (LRP5), mevalonate pyrophosphate decarboxylase (MVD), and lipocalin-2 (LCN2).
The amplification/qPCR employs primer pairs and probes selected from defined SEQ ID NO ranges for the converted genomic regions of CD3gamma/delta, CD4, CD8beta, LRP5, MVD, and LCN2. The detected methylation-discriminating signals enable identification of immune cell subsets based on demethylation at CpG sites in the marker amplicons. Demethylation is described as indicating specific immune cell types, including T-cell subsets, neutrophils, monocytes, NK cells, and B cells, with CD3gamma/delta and optionally CD3 epsilon used in the marker panel.
The document further describes a multi-level normalization and quantification strategy intended to support both relative and absolute cell-per-volume estimation. The strategy includes use of a housekeeping gene signal (e.g., GAPDH), an in silico bisulfite-converted GAP[GC] construct (CpG→GpC inversion) as a demethylation standard gene, and a calibrator plasmid containing equimolar unmethylated amplicon sequences. A spike-in GAP[GC] plasmid is described for absolute cell-per-volume estimation, and normalization concepts are presented in the context of genomic normaliser of methylation (GNoMs) and locus-specific and universal relative/universal and definitive unmethylation terms.
Claims Coverage
The document includes one independent claim covering an amplicon panel production and detection method, with additional inventive limitations supplied by dependent claims that refine sample context, add additional methylation targets, and specify calibration/standardization components and immune-cell subset readouts.
TpG-template bisulfite conversion qPCR amplicon panel for immune-cell markers
A method comprising bisulfite treating isolated genomic DNA from a human blood cell sample containing immune cells to convert unmethylated cytosines into uracil; amplifying the bisulfite treated DNA to produce a panel of amplicons; wherein amplifying comprises amplification and/or qPCR using a thymidine-cytosine (TpG) template carrying targets of converted genomic regions of CD3gamma/delta, CD4, CD8beta, LRP5, MVD, and LCN2 and primer pairs and probes selected from defined SEQ ID NO ranges for the converted genomic regions of CD3gamma/delta, CD4, CD8beta, LRP5, MVD, and LCN2; and detecting the panel of amplicons.
Newborn blood cell sample context
The method further includes obtaining the human blood cell sample from a newborn.
CD3 epsilon methylation-status analysis
The method further includes analyzing the methylation status of an amplicon for CD3 epsilon.
In silico bisulfite-converted GAP[GC] demethylation standard with CpG→GpC inversion
The method further uses an in silico bisulfite-converted recombinant nucleic acid containing a GAP[GC] construct that has all CpG dinucleotides inverted to GpC in the demethylation standard gene.
Calibrator plasmid with equimolar/unconverted un-methylated amplicon sequences
The method further includes using a calibrator plasmid that contains one copy of each amplicon sequence in its unconverted genomic state.
Immune cell subset quantification from detected amplicon panel
The method further includes quantifying specific immune cell types (CD4+ T cells, CD8+ T cells, B cells, natural killer (NK) cells, and neutrophils) as part of detecting a panel of amplicons.
Overall claim coverage centers on bisulfite treated genomic DNA from human blood immune-cell samples, TpG-template amplification/qPCR to produce and detect a marker amplicon panel for CD3gamma/delta, CD4, CD8beta, LRP5, MVD, and LCN2 using specified SEQ ID NO primer/probe sets, and downstream dependent refinements that add newborn context, additional methylation targets (CD3 epsilon), and defined demethylation/calibration standards and immune-cell subset quantification.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Diagnosing immune-related diseases and primary immunodeficiencies (PID).
Newborn screening using dried blood spots (DBS).
Detecting primary immunodeficiencies including SCID, X-linked agammaglobulinemia (XLA), and severe congenital neutropenia (SCN) with reported concordance to flow cytometry.
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