Genotyping An Autoimmune-Associated SIRPg SNP From Archived Oral Biopsy FFPE Tissues

Abstract

<h3>Introduction</h3> T-cells play critical roles in many autoimmune conditions, such as type I diabetes mellitus (T1D). They are also thought to play a significant role in many immune-mediated disorders affecting the oral soft tissues: lichen planus, graft vs. host disease, Sjogren syndrome. T-cells exert their functions via direct recognition through molecules expressed on the cell surface and secretion of factors that drive or suppress inflammatory responses locally. Signal regulatory protein gamma (SIRPg) is an immunomodulatory protein that is uniquely expressed on the cell surface of human T-cells. Our laboratory has shown that a T1D-linked genetic variant of the gene for SIRPg (rs2281808) correlates with reduced expression of SIRPg on the T-cell surface and that T-cells with less SIRPg surface expression produce more inflammatory molecules. We hypothesize that single nucleotide changes within the gene for SIRPg can lead to a predisposition for the development of immune-mediated pathosis. Our objective for this study was to assess the feasibility of genotyping individuals at single nucleotide polymorphism (SNP) rs2281808 from genomic DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue blocks. <h3>Materials and Methods</h3> Archived, de-identified formalin-fixed paraffin-embedded tissue blocks from oral soft tissue biopsies were used in this study. The tissue blocks ranged from 1-18 years of age. Three 5-mm thick sections per sample were deparaffinized with subsequent genomic DNA extraction using the QIAamp® DNA FFPE Tissue Kit and Deparaffinization Solution. DNA was quantitated using UV spectroscopy at 260 nm. Allelic discrimination PCR for rs2281808 genotyping was done using a TaqMan® assay and probes. <h3>Results</h3> Allelic discrimination PCR for rs2281808 was successful in ∼77% (46/60) of specimens tested. <h3>Conclusions</h3> This pilot study supports the feasibility of our methodology, however, further studies are necessary to assess the accuracy of the genotyping results obtained via this method.