Validation of Treg-signatures panel using targeted bisulfite NGS and Methylation Sensitive, High-Resolution Melting 3878

Abstract Description The FOXP3 gene, located on the X chromosome, encodes a transcription factor essential for regulatory T (Treg) cells, which maintain immunological self-tolerance and homeostasis. FOXP3 expression is tightly regulated by DNA methylation. Here, we have validated a multiplex PCR targeted NGS panel to analyze the methylation status of FOXP3 and 19 additional genes crucial to identity, stability, and function. Comprehensive validation was conducted by a blinded set of samples run through our NGS process for a total of seven times, while also assessing the lower limit of detection and quantification. This panel includes 29 assays and 141 CpG sites and was also assessed using Methylation-Sensitive High-Resolution Melting (MS-HRM) to assess accuracy and consistency with traditional NGS methods. MS-HRM offers a faster and more cost-effective approach by analyzing methylation status through melting temperature differences after bisulfite conversion, providing amplicon-wide data instead of single-CpG site resolution. Methylation concordance of the full panel was assessed, and Bland-Altman plots were generated to confirm equivalency between methods under two standard deviations. While reducing methylation resolution compared to NGS, MS-HRM is particularly suited for pilot studies or broader assessments where high-resolution data are unnecessary. For instance, a threshold of over 20% methylation of the FOXP3 TSDR indicates silencing, offering a useful benchmark for Treg-related research. Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)