Development and Validation of Real-Time PCR-Based TPMT Genotyping Method for Dose Adjustment of Thiopurine Compounds

Abstract

TPMT (thiopurine S-methyltransferase) methylates and, thus, inactivates thiopurine drugs such as azathioprine, 6- mercaptopurine, and 6-thioguanine, which are widely used to treat inflammatory bowel disease and childhood acute lymphoblastic leukemia. Genetic alterations in the TPMT gene directly affect the metabolism of such drugs, causing side effects including bone marrow toxicity, hepatotoxicity, and pancreatitis. PCR direct sequencing is the “gold standard” for TPMT genotyping, but it is time-consuming and expensive. In the Korean population, mostly ∗3C variants (c.719A>G) have been reported with incidence of about 0.3% to 7.6%; therefore, full sequencing is not economical for TPMT genotyping. We developed and validated a real-time PCR-based method for TPMT genotyping. SNP genotyping methods were developed under the real-time PCR platform using allele-specific primers and 5' nuclease probes. Three important SNPs for TPMT gene (c.238G>C, c.460G>C, and c.719A>G) were genotyped based on the differences in Ct (cycle threshold) in the real-time PCR system. Each reaction was confirmed by PCR direct sequencing. In addition, an automatic interpretation algorithm was developed to display genotypes at each site. The validity of amplification reactions was assessed based on the difference in Ct (DCt) for the reactions targeting 2 alleles at each SNP site. For each SNP site, homozygote specimens showed DCt greater than 2.0 and heterozygote specimens showed DCt smaller than 2.0. All these results were concordant with those confirmed by PCR direct sequencing. Overall turnaround time was around 2 hours, and 24 genomic DNA specimens could be analyzed simultaneously. We developed a real-time PCR-based genotyping kit for TPMT using allele-specific primers and probes, which produced consistent results with those by direct sequencing. This method is expected to be practically applied in clinical practice to determine the individualized dose of thiopurine compounds.