A Simple Method for TPMT and ITPA Genotyping Using Multiplex HRMA for Patients Treated with Thiopurine Drugs

Abstract

Thiopurine methyltransferase (TPMT) and inosine triphosphatase (ITPA) are crucial enzymes 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 enzymes correlates with the genetic polymorphism of the TPMT and ITPA genes, respectively, 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. In the ITPA gene, a c.94C>A mutation is significantly associated with an adverse thiopurine reaction. The aim of this study was to develop a quick and highly sensitive method for determining major TPMT and ITPA alleles. Here we present the molecular test for genotyping c.238G>C, c.460G>A, c.719A>G and c.94C>A changes based on multiplex high resolution melting analysis (HRMA). We analyzed DNA samples from 100 clinically diagnosed IBD patients treated with thiopurine drugs, and a known genotype in the positions 238, 460 and 719 of the TPMT gene as well as in position 94 of the ITPA gene. Our results obtained with multiplex HRMA indicated 100 % accuracy in comparison with data from restriction fragments length polymorphism (RFLP) and standard DNA sequencing. We conclude, that multiplex HRMA can be used as a quick, sensitive and efficient alternative diagnostic method compared to conventional techniques for the determination of TPMT*2, TPMT*3A and TPMT*3C alleles and c.94C>A change in the ITPA gene.

Fig. 1

Multiplex high resolution melting (HRM) analysis for exon 6 and 9 of the TPMT gene. a Melting profile of both polymerase chain reaction (PCR) products. b Normalized difference plots of amplicons corresponding to exon 9 (c.719). c Normalized difference plots of amplicons corresponding to exon 6 (c.460 and additionally locus c.474)

Fig. 2

Multiplex high resolution melting analysis (HRMA) for exon 4 of the TPMT gene and exon 2 of the ITPA gene. a Melting profile of both polymerase chain reaction (PCR) products. b Normalized difference plots of amplicons corresponding to exon 4 (c.238). c Normalized difference plots of amplicons corresponding to exon 2 (c.94)