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Updated: Apr 26, 2026
Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry
Published on: June 21, 2018
Development of a rapid clinical TPMT genotyping assay
Paul R Burchard1, Ahmad N Abou Tayoun1, Joel A Lefferts2
1Department of Pathology, The Geisel School of Medicine at Dartmouth, Hanover, NH, USA; Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA.
Objectives:
Thiopurine compounds are commonly used in the treatment of childhood acute lymphoblastic leukemia, and as immunosuppressants following organ transplantation or for treatment of various autoimmune disorders. Thiopurine S-methyltransferase (TPMT) is required for detoxification, through S-methylation, of 6-thioguanine nucleotides (TGNs), a byproduct of thiopurine metabolism. Single nucleotide polymorphisms (SNPs) in the TPMT gene have been shown to affect its function, with some variants associated with serious clinical manifestations including severe to fatal myelosuppression and organ transplant rejection following treatment with standard thiopurine doses. In this study, we describe a TaqMan real time PCR allelic discrimination assay requiring minimal DNA input for TPMT genotyping.
Design And Methods:
We designed controls for the homozygous wild type and allelic variants of TPMT*2, *3B, and *3C. Genomic DNA was extracted from an additional 412 human blood samples. The samples were tested for the TPMT*2, *3B, *3C, and *3A polymorphisms by TaqMan genotyping assays using the AB 7500 FAST Real-Time PCR instrument. Allelic discrimination plots were used to identify each mutation.
Results:
The TaqMan assay correctly genotyped all custom control DNA samples. Of the 412 tested samples, our assay identified 375 samples as wild-type *1/*1 (91.02%), 3 as *1/*2 (0.73%), 1 as *1/*3B (0.24%), 3 as *1/*3C (0.73%), 27 presumed to be *1/*3A (6.55%), and 3 as *3B/*3A (0.73%).
Conclusions:
The clinical implications of TPMT genotyping, along with the simplicity and specificity of the TaqMan genotyping assays make this test highly suitable for use in a clinical laboratory.
