B-315 Analytical validation and implementation of a high-throughput allele-specific qPCR assay for the PAI-1 4G/5G polymorphism genotyping

Abstract

Routine genotyping of common variants in many laboratories still relies on labor-intensive methods with limited throughput. Recent advances in qPCR chemistry (e.g., modified fluorescent probes) and automation (e.g., automated liquid handling) enable more efficient and scalable workflows for clinically relevant testing. Here, we developed and analytically validated a low-cost, high-throughput allele-specific qPCR assay using the SERPINE1 promoter 4G/5G insertion/deletion (rs1799762)—a major determinant of plasminogen activator inhibitor-1 (PAI-1) levels—as a clinical case study. The 4G allele has been associated with higher PAI-1 expression, impaired fibrinolysis, and increased risk of thrombotic and obstetric complications. We developed an allele-specific hydrolysis-probe qPCR assay using Minor Groove Binder (MGB)-modified probes targeting the SERPINE1 promoter 4G/5G insertion/deletion (rs1799762) and performed genotype calling by endpoint allelic discrimination. Probes were synthesized by Integrated DNA Technologies (IDT), and probe concentrations were systematically titrated to maximize genotype cluster separation and minimize overlap between homozygous and heterozygous calls. Analytical validation included 145 whole-blood samples previously genotyped by Sanger sequencing (reference method). Automated nucleic acid extraction and qPCR were performed on the Roche FLOW platform, with reactions run in duplicate. Agreement with Sanger was summarized as overall and by-genotype concordance. Call rate and cluster quality (i.e., well-defined, non-overlapping clusters) were assessed from endpoint plots. Signal-to-noise was summarized as the ratio of matched-probe to mismatched-probe maximum fluorescence (RFU). Precision was assessed using representative samples from all genotypes via intra-run repeatability (9 samples tested in triplicate within a single run) and inter-run repeatability (10 samples processed independently across three different days). Turnaround time was measured from extraction to genotype call in a single 96-well run containing 93 clinical samples. Overall concordance with Sanger was 100% (145/145), with a 100% call rate and no no-calls or ambiguous clusters across the validation cohort. By-genotype concordance was 100% for 4G/4G (22/22), 4G/5G (62/62), and 5G/5G (61/61). Endpoint allelic discrimination plots showed clear spatial separation of all three genotype clusters. Mean matched-probe maximum fluorescence (RFU) was approximately 5-fold higher than mismatched-probe signal, supporting a high signal-to-noise ratio for endpoint calling. Precision studies demonstrated 100% intra-run and inter-run reproducibility. Genotype frequencies were 15.17% (4G/4G), 42.76% (4G/5G), and 42.07% (5G/5G). Time-to-result was approximately 5 hours (extraction to genotype call) for a 93-sample run. We validated and implemented a rapid, allele-specific qPCR assay for SERPINE1 (PAI-1) 4G/5G genotyping, demonstrating 100% concordance with Sanger sequencing in 145 clinical whole-blood samples. The fully automated workflow is robust and scalable, reduces time-to-result, and improves laboratory efficiency. This approach provides a practical model for developing and implementing qPCR-based genotyping assays for other clinically relevant variants, enabling routine personalized testing to inform thrombotic risk assessment in clinical practice.