RT-PCR permits simultaneous genotyping of thiopurine S-methyltransferase allelic variants by multiplex induced heteroduplex analysis
✍ Scribed by Nigel Wood; Adrian Fraser; Jeffrey Bidwell; Graham Standen
- Publisher
- John Wiley and Sons
- Year
- 2004
- Tongue
- English
- Weight
- 213 KB
- Volume
- 24
- Category
- Article
- ISSN
- 1059-7794
No coin nor oath required. For personal study only.
✦ Synopsis
Communicated by Linda Tyfield
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.460G4A and c.719A4G, 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.719A4G allelic variant, TPMT*3C, as well as the silent alteration c.474T4C. 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.