title: RT-PCR permits simultaneous genotyping of thiopurine S-methyltransferase allelic variants by multiplex induced heteroduplex analysis.
abstract: 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.
authors.affiliation: Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, UK. nigel.wood@bristol.ac.uk
authors.forename: Nigel
authors.initials: N
authors.lastname: Wood
authors.forename: Adrian
authors.initials: A
authors.lastname: Fraser
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Bidwell
authors.forename: Graham
authors.initials: G
authors.lastname: Standen
mesh_terms: D000483:Alleles; D018076:DNA, Complementary / Q000235:genetics; D014644:Genetic Variation / Q000235:genetics*; D005838:Genotype; D006402:Hematologic Diseases / Q000235:genetics; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D008780:Methyltransferases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D012313:RNA / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D012313:RNA; D008780:Methyltransferases; C022745:thiopurine methyltransferase