Development of protocol for detecting cytochrome p450 genes related to drug hypersensitivity by cold-pcr in comparison with target gene sequencing results

Abstract

In precision medicine era, multiple factors are needed in clinical decision-making because of ethnic and racial genetic diversity, family history, and other health details. Although advanced techniques are evolved, there is still an obstacle to overwhelm pharmacogenetic (PGx) implementation in developing countries. The objective of this study is to provide easy-to-use method that roughly estimate the patient’s carrier type, easily prescreen out normal ones before sequencing or genotyping usage for actual genetic status. We developed fast co-amplification at lower denaturation temperature (COLD)-PCR to differentiate genetic variant non-carriers from carriers. Cytochrome P450 (CYP) enzymes play oxidative biotransformation of drugs. Of all the CYPs, CYP2C9, CYP2C19, and CYP2D6 isoforms highly involve in PGx testing. Therefore, five variants related to these CYPs focusing on Thai and Asian populations were selected. The optimal annealing temperature and critical denaturation temperature (Tc) were determined and evaluated using Sanger sequencing of 27 randomly collected samples. The single-reaction issue was resolved in combination of precise Tc including Tc1 = 75.0 °C (10 cycles), Tc2 = 87.0 °C (10 cycles), and, finally, Tc3 = 90.5 °C (20 cycles). The results showed 100% sensitivity and specificity, with perfect agreement (κ = 1.0) compared to Sanger sequencing. Moreover, this study also introduces whole exome sequencing (WES) on two samples to compare previously developed results from COLD-PCR with validated method - Sanger sequencing. Results showed perfect correlation between three methods. Although, the efficiency and reliability become increased using sequencing technique, some limitations still exist complementing in routine medical laboratory workflow including high cost and time consuming. Therefore, by using newly developed fast-COLD-PCR as the screening test, it can be an alternative pipeline for pharmacogenomics profiling. It benefits cost and time saving by screening out wild-type samples and only positive mutant samples need to perform genotyping analysis in further.