Introduction of new alternative pipeline using multiplexed fast COLD‑PCR together with sequencing approach...

In precision medicine, multiple factors are involved in clinical decision-making because of ethnic and racial genetic diversity, family history and other health factors. Although advanced techniques have evolved, there is still an economic obstacle to pharmacogenetic (PGx) implementation in developing countries. The aim of the present study was to provide an alternative pipeline that roughly estimate patient carrier type and prescreen out wild-type samples before sequencing or genotyping to determine genetic status. Fast co-amplification at lower denaturation temperature (COLD)-PCR was used to differentiate genetic variant non-carriers from carriers. The majority of drugs are hepatically cleared by cytochrome P450 (CYP) enzymes and genes encoding CYP enzymes are highly variable. Of all the CYPs, CYP2 family of CYP2C9, CYP2C19, and CYP2D6 isoforms have clinically significant impact on drugs of PGx testing. Therefore, five variants associated with these CYPs were selected for preliminary testing with this novel pipeline. For fast COLD-PCR, the optimal annealing temperature and critical denaturation temperature were determined and evaluated via Sanger sequencing of 27 randomly collected samples. According to precise Tc, to perform in a single-reaction is difficult. However, in this study, this issue was resolved by combination of precise Tc using 10+10+20 cycles. The results showed 100% sensitivity and specificity, with perfect agreement (κ=1.0) compared with Sanger sequencing. The present study provides a prescreening platform by introducing multiplex fast COLD-PCR as a pharmacoeconomic implementation. Our study just present in five variants which are not enough to describe patient metabolic status. Therefore, other actional genetic variants are still needed to cover the actual patient's genotypes. Nevertheless, the proposed method can well-present its efficiency and reliability for serving as a PGx budget platform in the future.