Genotyping Of Angiotensin-related Targets In Hypertension Pharmacogenomics

Objective: This study aims to develop a multiplex allele-specific quantitative PCR(AS-q PCR)analysis as a hypertension pharmacogenomics tool,which can enable discrimination of RAAS(renin-angiotensin-aldosterone system)-associated genotypes(ACE(I/D),CYP2C9*3 and AGTR1(1166A>C))affecting the drug efficacy in anti-hypertension treatment.Methods: Employing polymerase producer suggested amplification assay as a basic reaction,three rounds of F-primer screening/validation were conducted:(i)The reciprocal second,third and fifth base mismatch were introduced at the 3’-terminal of allele-specific F-primers that identified CYP2C9*3 and AGTR1(1166A>C)nucleotide polymorphism,respectively.These F-primers were screened to enable maximization of?Cq(difference in threshold cycle between the wild-type F-primer amplification assay and the mutant F-primer amplification assay)by using synthesized plasmid model and a uniplex analysis;(ii)A series of concentrations of genomic DNA from human oral swab cells was prepared,and a calibration curve was established by detection of genomic DNA using triplex AS-q PCR analysis.Then selected F-primers contained in amplification analysis were optimized by amplification efficiency;(iii)203 samples of genomic DNA from human oral swab cells were collected.And agreement analysis was executed considering NGS as reference method.In addition,performance of proposed triplex AS-q PCR was evaluated with cross-reactivity,interfering experiments,reproducibility and stability testing.Results: After three rounds of F-primers screening/validation and performance evaluation,a triplex AS-q PCR analysis was successfully established,which can accurately discriminate the genotypes of ACE(I/D),CYP2C9*3 and AGTR1(1166A>C).The calibration curves indicated that amplification efficiencies ranged from0.9 to 1.1(R2>0.99).And analytical sensitivity appeared at least 1.25 ng.There was no statistically significant difference between the analysis and NGS.Cross-reactivity showed that homologous genes were observed not to cross react.The interfering test displayed the template DNA mixed with Streptococcus salivarius subsp.salivarius,haemoglobin,toothpaste,and tar nicotine base,etc,did not affect the interpretation of genotyping.Reproducibility assessment exhibited that intra-batch coefficient of variation(CV)was less than 1%,and inter-batch CV less than 2%.Results from stability experiments also demonstrated well stability.Conclusion: As a hypertension pharmacogenomics tool,the triplex AS-q PCR analysis described in this study can accurately determine RAAS-associated genotypes(ACE(I/D),CYP2C9*3 and AGTR1(1166A>C))guiding anti-hypertension drug delivery.