RT-PCR permits simultaneous genotyping of thiopurine S-methyltransferase allelic variants by multiplex induced heteroduplex analysis^†

Thiopurine-based drugs are a widely prescribed group of medications. Their tolerance and effectiveness is dependent on an individual's ability to metabolize these compounds. An essential enzyme for the metabolism of these drugs is thiopurine S-methyltransferase (TPMT), whose activity is subject to genetic variation. Genotyping of the most frequent allelic variants in TPMT affords an extremely accurate prediction of the three clinical phenotypes: high, intermediate, and low enzyme activity. One constraint of most genotyping methods is the inability to demonstrate physical linkage between two sequence variants that occur in different exons, e.g., c.460G>A and c.719A>G, which give rise to TPMT^*3, the most common defective allele in Caucasian populations. Using mRNA/cDNA as a template enables analysis of both sequence variants in a single assay. This approach could be applicable to other genes where allelic variation (in-cis and in-trans) is due to alterations in different exons. Induced heteroduplex generator analysis has previously been shown to discriminate in-cis and has also been suitable for multiplexing. In this method we have exploited both these features and for the first time have applied them to a RT-PCR analysis. The primary reagent developed allows unequivocal resolution of TPMT^*3A and the alleles carrying the c.719A>G allelic variant, TPMT^*3C, as well as the silent alteration c.474T>C. The TPMT^*3B variant has not been observed. A secondary reagent, which can be multiplexed, identifies the TPMT^*2 allele. Hum Mutat 24:93–99, 2004. © 2004 Wiley-Liss, Inc.