Scorpion primer PCR analysis for genotyping of allele variants of thiopurine s‑methyltransferase*3.

Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurines. Mutations in the TPMT gene can affect drug activity, which may have adverse effects in humans. Thus, genotyping can help elucidate genetic determinants of drug response to thiopurines and optimize the selection of drug therapies for individual patients, effectively avoiding palindromia during maintenance treatment caused by insufficient dosing and the serious side effects caused by excessive doses. The current available detection methods used for TPMT*3B and TPMT*3C are complex, costly and time‑consuming. Therefore, innovative detection methods for TPMT genotyping are urgently required. The aim of the present study was to establish and optimize a simple, specific and timesaving TPMT genotyping method. Using the principles of Web‑based Allele‑Specific PCR and competitive real‑time fluorescent allele‑specific PCR (CRAS‑PCR), two pairs of Scorpion primers were designed for the detection of TPMT*3B and *3C, respectively, and a mutation in TPMT*3A was inferred based on data from TPMT*3B and *3C. In total, 226 samples from volunteers living in Chongqing were used for CRAS‑PCR to detect TPMT*3 mutations. Results showed that nine (3.98%) were mutant (MT) heterozygotes and none were MT homozygotes for TPMT*3C, and no TPMT*3A and TPMT*3B mutations were found. Three TPMT*3C MT heterozygotes were randomly selected for DNA sequencing, and CRAS‑PCR results were consistent with the sequencing results. In conclusion, in order to improve simplicity, specificity and efficiency, the present study established and optimized CRAS‑PCR assays for commonly found mutant alleles of TPMT*3A (G460A and A719G), TPMT*3B (G460A), and TPMT*3C (A719G).