Genotyping Protocol for Identifying Genetic Risk Markers Associated with Alpha-Gal Syndrome.

Alpha-gal syndrome (AGS) is an IgE-mediated allergy to galactose-α-1,3-galactose (α-Gal), typically triggered by tick bites. Genetic factors, including human leucocyte antigen (HLA) class II polymorphisms and selected single nucleotide polymorphisms (SNPs) in immune and glycan-related genes, are hypothesized to modulate susceptibility and clinical expression. Here we provide a step-by-step protocol to genotype risk markers from ethylenediaminetetetraacetic acid (EDTA) whole blood sample, combining (i) manual DNA extraction (Qiagen), (ii) SNP genotyping by a real-time/quantitative PCR (qPCR) method (FluoQube system), and (iii) HLA class I/II genotyping by sequence-specific oligonucleotide (SSO method using a Luminex® xMAP system). The protocol details reagent preparation, thermal cycling and hybridization conditions, data analysis, quality control (QC), and reporting. We also include troubleshooting notes, acceptance criteria, and guidance for clinical interpretation (e.g., integrating genotypes with α-Gal sIgE and clinical phenotype). This protocol enables reproducible implementation of genetic-risk workflows for AGS in molecular diagnostics or translational-research settings.