Thiopurine S-methyltransferase pharmacogenetics: variant allele functional and comparative genomics

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.

Thiopurine S-methyltransferase pharmacogenetics: insights, challenges and future directions

“…This phenomenon has also been observed for many other common genetic polymorphisms that alter only a single amino acid (Freimuth et al, 2001;Thomae et al, 2002;Adjei et al, 2003;Thomae et al, 2003;Weinshilboum and Wang, 2004a;Ji et al, 2005). Of the allozymes studied by Salavaggione et al (2005), TPMT*3A, *3B, *3C and *2 displayed the most striking effects. This phenomenon was first observed a decade ago when TPMT*3A, *3B and *3C were initially described (Szumlanski et al, 1996).…”

“…The reason why altering only two amino acids out of 245 results in such a striking phenotype will be addressed subsequently. TPMT*3C and the first variant allele identified, TPMT*2, do not result in such dramatic decreases in levels of enzyme protein as do *3A and *3B, but they are also associated with significant decreases in quantity of TPMT protein (Tai et al, , 1999Wang et al, 2003;Salavaggione et al, 2005). On the basis of population studies, *3A and *3C are the predominant variant alleles, with *2 contributing to a lesser extent.…”

Pharmacogenomics and personalized medicine: a review focused on their application in the Chinese population

“…The TPMT gene is highly polymorphic; more than 25 variants have been identified. Four alleles (TPMT*2, *3A, *3B, and *3C) account for approximately 95% of inherited TPMT deficiency [109][110][111] . The wild-type allele, TPMT*1, encodes the fully active enzyme, and TPMT*2, TPMT*3A and TPMT*3C are the most prevalent genotypes in Caucasians, together accounting for 80% to 95% of the polymorphic alleles that lead to a significant reduction in enzyme activity due to enhanced rates of proteolysis of the mutant proteins [112] .…”

“…TPMT is a cytosolic methylating enzyme whose physiological role remains unclear despite extensive investigation. It is reported that a reduction in TPMT activity, caused by genetic polymorphisms results in severe and hematological toxicity in patients treated with standard doses of thiopurines [108,109] . Approximately 0.3% of individuals in a general population had low levels of TPMT activity, with approximately 10% of individuals expressing intermediate levels [108] .…”

Clinical Practice Recommendations for the Management and Prevention of Cisplatin-Induced Hearing Loss Using Pharmacogenetic Markers

“…In African populations, *3C and *2 are also common. All of these variants demonstrate reduced in vitro enzyme activity 49 . Around 10% of the population Copyright Ó 2016 Wolters Kluwer Health, Inc.…”