Development of an Assay to Distinguish Between Genotypes of the Leucyl-Cystinyl Aminopeptidase Gene

Abstract

Introduction: The leucyl-cystinyl aminopeptidase gene rs4869317 single nucleotide polymorphism (SNP) is associated with multiple conditions; for example, patients with the rs4869317 TT genotype experiencing septic shock have increased plasma clearance of vasopressin versus AA or AT genotypes. The current aim was to assess the appropriateness of a TaqMan? SNP genotyping assay as a tool to identify the rs4869317 SNP in human samples, given its association with serious medical conditions. Methods: Genomic DNA was amplified using sequence-specific forward/reverse primers, and allelic discrimination was achieved using VIC?- or FAM?-labeled TaqMan probes. The Applied BiosystemsTM QuantStudioTM 12?K Flex Real-Time PCR System was used for SNP genotyping, and data were analyzed by the TaqMan Genotyper software. Results: Intra- and interassay precisions were established, with results consistent within and between genotyping assays and between instruments and operators. Assay robustness was established across a range of input DNA (11?56?ng). The assay was 100% accurate for discriminating between alleles of the rs4869317 SNP (30 samples) and was concordant with results from independently performed Sanger sequencing. The detection of leucyl-cystinyl aminopeptidase rs4869317 SNP using TaqMan genotyping methods was shown to be an appropriate, reliable, and rapid analytical tool for generating allelic calls. Conclusion: This assay is a valid tool for genotyping analyses in clinical trials and potential applications in precision medicine.

Keywords

• single nucleotide polymorphisms
• TaqMan
• genotyping techniques
• leucyl aminopeptidase
• rs4869317
• vasopressin