The major inactivating mutations at the human TPMT locus have been identified and can be reliably detected by PCR-based methods, which show an excellent concordance between genotype and phenotype. The detection of TPMT mutations provides a molecular diagnostic method for prospectively identifying TPMT-deficient and heterozygous patients.

TPMT is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including medications such as mercaptopurine and thioguanine. TPMT activity exhibits autosomal codominant genetic polymorphism, and patients inheriting TPMT deficiency are at high risk of potentially fatal hematopoietic toxicity. The most prevalent mutant alleles associated with TPMT deficiency in humans have been cloned and characterized (TPMT‫2ء‬ and TPMT‫3ء‬A), but the mechanisms for loss of catalytic activity have not been elucidated. In the present study, we established that erythrocyte TPMT activity was significantly related to the amount of TPMT protein on Western blots of erythrocytes from patients with TPMT activities of 0.4-23 units͞ml pRBC (r s ‫؍‬ 0.99; P < 0.001). Similarly, heterologous expression of wild-type (TPMT‫)1ء‬ and mutant (TPMT‫2ء‬ and TPMT‫3ء‬A) human cDNAs in yeast and COS-1 cells demonstrated comparable levels of TPMT mRNA but significantly lower TPMT protein with the mutant cDNAs. Rates of protein synthesis were comparable for wild-type and mutant proteins expressed in yeast and with in vitro translation in rabbit reticulocyte lysates. In contrast, pulse-chase experiments revealed significantly shorter degradation halflives for TPMT‫2ء‬ and TPMT‫3ء‬A (ϳ0.25 hr) compared with wild-type TPMT‫1ء‬ (18 hr). The degradation of mutant proteins was impaired by ATP depletion and in yeast with mutant proteasomes (pre-1 strain) but unaffected by the lysosomal inhibitor chloroquine. These studies establish enhanced degradation of TPMT proteins encoded by TPMT‫2ء‬ and TPMT‫3ء‬A as mechanisms for lower TPMT protein and catalytic activity inherited by the predominant mutant alleles at the human TPMT locus.TPMT (S-adenosyl L-methionine:thiopurine S-methyltransferase; EC 2.1.1.67) is a cytosolic protein that catalyzes the S-methylation of mercaptopurine, mercaptopurine formed from the prodrug azathioprine, 6-thioguanine, and other aromatic and heterocyclic sulfhydryl compounds. TPMT activity exhibits codominant polymorphism in Caucasian and AfricanAmerican populations (1, 2), with about 10% having intermediate activity due to heterozygosity at the TPMT locus and approximately 1 in 300 inheriting TPMT deficiency. Decreased S-methylation in TPMT-deficient and heterozygous patients leads to more extensive accumulation of thioguanine nucleotides (3), the principal cytotoxic metabolites (4), thereby producing greater toxicity. Patients inheriting TPMT deficiency have a pronounced risk of potentially life-threatening hematopoietic toxicity when treated with conventional doses of these medications (5-8), and patients with heterozygosity at the TPMT locus have lower activity and an intermediate risk of toxicity.The inherited polymorphism of TPMT activity in humans has now been elucidated at the molecular level with the identification of inactivating mutations in the human TPMT gene (9, 10). The initial molecular defect to be discovered was a single G238 3 C transversion mutation leading to an...

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