Epigenetic method to detect and distinguish IPEX and IPEX-like syndromes, in particular in newborns
Inventors
Assignees
Precision for Medicine GmbH • Board Of Truees Of Leland Stanford Junior University • Leland Stanford Junior University
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Publication Number
US-12247258-B2
Publication Date
2025-03-11
Expiration Date
Abstract
The present invention relates to an epigenetic method for identifying IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked) syndrome and/or IPEX-like syndrome in a human subject using the methylation status of F0XP3 and a control gene specific for a CD4 and/or CD3 T cell. The method is used to distinguish IPEX from IPEX-like and from IPEX-unrelated disorders.
Core Innovation
The invention provides an epigenetic qPCR approach for newborn and early-life diagnosis of IPEX and IPEX-like syndromes based on bisulfite-converted methylation status of FOXP3. It uses the FOXP3 Treg-specific demethylated region (TSDR) to determine CpG demethylation in a human subject, with analysis of FOXP3 demethylation in combination with a control T-cell gene region (CD4 and/or CD3) normalized to an unmethylated housekeeping gene region (GAPDH).
Diagnosis is produced from quantitative demethylation levels or ratios using defined reference ranges. The document reports that non-IPEX Treg disorder is identified when demethylation is below about 95–99%, IPEX-like syndrome is identified when demethylation is within about 95–99%, and IPEX is identified when demethylation is above about 99%. The approach is framed as an epigenetic qPCR readout that distinguishes immune cell/Treg deficiencies relevant to IPEX and IPEX-like disease.
The document describes rationale and implementation using dried blood spots (DBS), including Guthrie cards. It further provides a normalization and quantification strategy using calibration plasmids and an artificial GAP[GC] nucleic acid template with inverted CpG dinucleotides, and reports concordance with flow cytometry for identifying immune cell/Treg deficiencies relevant to IPEX and IPEX-like disease.
The document also describes kit and treatment/monitoring embodiments based on the methylation and immune quantification outputs, including treating IPEX syndrome and/or IPEX-like syndrome based on demethylation criteria measured from the FOXP3 amplicons and kit embodiments for bisulfite reagent and methylation analysis materials.
Claims Coverage
The partial content includes two independent claims. Across these independent claims, the inventive coverage centers on producing and detecting a panel of amplicons from bisulfite-treated genomic DNA for FOXP3, CD4, CD3, and a housekeeping gene with a GAP[GC] artificial region, and using measured demethylation thresholds to stratify and trigger treatment for IPEX versus IPEX-like syndrome.
Bisulfite treatment and amplicon panel production for FOXP3/CD4/CD3/housekeeping
Bisulfite treating isolated genomic DNA from a cell sample of a human subject to convert unmethylated cytosines into uracils; producing the panel of amplicons by amplifying from the bisulfite treated DNA a human gene region for FOXP3, a human CD4 gene region, a human CD3 gene region, and a housekeeping gene region; and detecting the panel of amplicons.
Housekeeping calibration using GAP[GC] artificial CpG-inverted region
Amplifying a nucleic acid template comprising an unconverted glyceraldehyde 3-phosphate dehydrogenase (GAPDH) artificial region with inverted CpG dinucleotides (GAP[GC]) as part of producing the panel of amplicons.
Demethylation-triggered treatment stratification for IPEX and IPEX-like
Treating IPEX syndrome when the amplicons for the human gene region for FOXP3 show demethylation of at least one CpG position above 99%, and treating IPEX-like syndrome when the amplicons for the human gene region for FOXP3 show demethylation of at least one CpG position between 95% and 99%.
Overall, the claims coverage supports a unified workflow where bisulfite-treated genomic DNA is used to produce and detect a FOXP3-focused amplicon panel with accompanying CD4/CD3 and housekeeping components, including an artificial GAP[GC] region, and where measured FOXP3 CpG demethylation thresholds differentiate IPEX from IPEX-like syndrome to trigger treatment.
Stated Advantages
Enables newborn and early-life diagnosis of IPEX and IPEX-like syndromes using epigenetic qPCR based on FOXP3 methylation status.
Provides quantification and concordance with flow cytometry for identifying immune cell/Treg deficiencies relevant to IPEX and IPEX-like disease.
Uses dried blood spots (DBS) to support the diagnostic approach in newborn and early-life settings.
Supports kit and treatment/monitoring embodiments based on methylation and immune quantification outputs.
Documented Applications
Newborn and early-life diagnosis of IPEX and IPEX-like syndromes using bisulfite-converted methylation status of FOXP3, including TSDR, with control normalization to an unmethylated housekeeping gene region.
Treating IPEX syndrome and/or IPEX-like syndrome in a human subject based on FOXP3 CpG demethylation measured from the amplicons.
Using dried blood spots (DBS) and Guthrie cards as sample types for the methylation analysis.
Kit embodiments for bisulfite reagent and methylation analysis materials.
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