Development and Validation of Multiplex Real-Time PCR Assays for Allele Discrimination and Genotyping of ABCG2 Q126X and Q141K

Gout is a metabolic disorder caused by chronic hyperuricemia, which leads to monosodium urate crystal deposition and subsequent inflammatory arthritis. Genetic factors, particularly those regulating urate transport, play a major role in susceptibility to hyperuricemia and gout. Among these, the functional ABCG2 variants Q126X (rs72552713) and Q141K (rs2231142) are well-established genetic determinants of impaired urate excretion. In this study, we developed a hydrolysis probe-based multiplex real-time polymerase chain reaction (PCR) assay for allele discrimination and genotyping of the ABCG2 Q126X and Q141K variants in separate reactions. This assay demonstrated high analytical specificity without cross-reactivity in both synthetic and clinical samples and maintained reliable genotype detection across clinically relevant DNA concentrations. In method comparison using 1653 clinical samples, the multiplex real-time PCR assay showed complete concordance with Sanger sequencing (Cohen’s κ = 1.00). Combined genotype analysis (Q126X/Q141K) demonstrated a gene dose-dependent association with serum uric acid levels and hyperuricemia risk, consistent with the estimated ABCG2 transport function. These findings indicate that the developed assay provides a reliable and practical molecular tool for rapid ABCG2 genotyping and genetic risk assessment of hyperuricemia and gout.