ORIGINAL ARTICLESThiopurine S-methyltransferase pharmacogenetics: variant allele functional and comparative genomicsSalavaggione, Oreste E.a; Wang, Lieweia; Wiepert, Mathieub; Yee, Vivien C.c; Weinshilboum, Richard M.aAuthor Information aDepartments of aMolecular Pharmacology and Experimental Therapeutics bHealth Sciences Research, Mayo Clinic College of Medicine-Mayo Clinic, Rochester, Minnesota cDepartment of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA Sponsorship: This study was supported in part by National Institutes of Health grants R01 GM28157 (O.E.S., L.W., R.M.W.), R01 GM35720 (O.E.S., L.W., R.M.W.) and U01 GM61388, The Pharmacogenetics Research Network (O.E.S., L.W., M.W., V.C.Y., R.M.W.). Correspondence and requests for reprints to Richard Weinshilboum, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Medical School-Mayo Clinic-Mayo Foundation, 200 First Street SW, Rochester, MN 55905, USA Tel: +1 507 284 2246; fax: +1 507 284 9111; e-mail: [email protected] Received 30 March 2005 Accepted 13 June 2005 Pharmacogenetics and Genomics: November 2005 - Volume 15 - Issue 11 - p 801-815 doi: 10.1097/01.fpc.0000174788.69991.6b Buy Metrics Abstract Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. Genetic polymorphisms for TPMT are a major factor responsible for large individual variations in thiopurine toxicity and therapeutic effect. The present study investigated the functional effects of human TPMT variant alleles that alter the encoded amino acid sequence of the enzyme, TPMT*2, *3A, *3B, *3C and *5 to *13. After expression in COS-1 cells and correction for transfection efficiency, allozymes encoded by these alleles displayed levels of activity that varied from virtually undetectable (*3A,*3B and *5) to 98% (*7) of that observed for the wild-type allele. Although some allozymes had significant elevations in apparent Km values for 6-mercaptopurine and S-adenosyl-L-methionine (i.e. the two cosubstrates for the reaction), the level of enzyme protein was the major factor responsible for variation in activity. Quantitative Western blot analysis demonstrated that the level of enzyme protein correlated closely with level of activity for all allozymes except TPMT*5. Furthermore, protein levels correlated with rates of TPMT degradation. TPMT amino acid sequences were then determined for 16 non-human mammalian species and those sequences (plus seven reported previously, including two nonmammalian vertebrate species) were used to determine amino acid sequence conservation. Most human TPMT variant allozymes had alterations of residues that were highly conserved during vertebrate evolution. Finally, a human TPMT homology structural model was created on the basis of a Pseudomonas structure (the only TPMT structure solved to this time), and the model was used to infer the functional consequences of variant allozyme amino acid sequence alterations. These studies indicate that a common mechanism responsible for alterations in the activity of variant TPMT allozymes involves alteration in the level of enzyme protein due, at least in part, to accelerated degradation. © 2005 Lippincott Williams & Wilkins, Inc.