Detection of thiopurine S-methyltranseferase mutations by multiplex PCR

Thiopurine methyltransferase (TPMT) is an enzyme involved in the metabolism of thiopurine drugs (azathioprine and 6-mercaptopurine) used in the treatment of leukemia or inflammatory bowel diseases (IBD). The activity in these enzyme correlates with the genetic polymorphism of the TPMT genes, which determines an individual reaction and dosing of thiopurines. Three main TPMT alleles: TPMT*2 (c.238G>C), TPMT*3A (c.460G>A, c.719A>G) and TPMT*3C (c.719A>G) account for 80-95 % of inherited TPMT deficiency in different populations in the world. The aim of this study was to use a quick and highly sensitive method for determining major TPMT alleles. Here we present the molecular test for genotyping c.238G>C, c.460G>A, c.719A>G changes based on multiplex high resolution melting analysis (HRMA). We analyzed 90 DNA samples from normal population a known genotype in the positions 238, 460 and 719 of the TPMT gene. Our results obtained with multiplex HRMA indicated 100 % accuracy in comparison with DNA sequencing. We conclude that multiplex HRMA can be used as a quick, sensitive and efficient diagnostic method compared to conventional techniques for the determination of TPMT*2, TPMT*3A and TPMT*3C alleles.