Thiopurine S-methyltransferase (TPMT) pharmacogenetics: variant allele functional genomics (original) (raw)
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Biochemical and Biophysical Research Communications, 2003
Human thiopurine S-methyltransferase (TPMT) is an enzyme responsible for the detoxification of widely used thiopurine drugs such as azathioprine (Aza). Its activity is inversely related to the risk of developing severe hematopoietic toxicity in certain patients treated with standard doses of thiopurines. DNA samples from four leucopenic patients treated with Aza were screened by PCR-SSCP analysis for mutations in the 10 exons of the TPMT gene. Four missense mutations comprising two novel mutations, A83T (TPMT*13, Glu 28 Val) and C374T (TPMT*12, Ser 125 Leu), and two previously described mutations, G430C (TPMT*10, Gly 144 Arg) and T681G (TPMT*7, His 227 Gln) were identified. Using a recombinant yeast expression system, kinetic parameters (K m and V max ) of 6-thioguanine S-methylation of the four TPMT variants were determined and compared to those obtained with wild-type TPMT. This functional analysis suggests that these rare allelic variants are defective TPMT alleles. The His 227 Gln variant retained only 10% of the intrinsic clearance value (V max /K m ratio) of the wild-type enzyme. The Ser 125 Leu and Gly 144 Arg variants were associated with a significant decrease in intrinsic clearance values, retaining about 30% of the wild-type enzyme, whereas the Glu 28 Val variant produced a more modest decrease (57% of the wild-type enzyme). The data suggest that the sporadic contribution of the rare Glu 28 Val, Ser 125 Leu, Gly 144 Arg, and His 227 Gln variants may account for the occurrence of altered metabolism of TPMT substrates. These findings improve our knowledge of the genetic basis of interindividual variability in TPMT activity and would enhance the efficiency of genotyping methods to predict patients at risk of inadequate responses to thiopurine therapy.
Dna and Cell Biology, 1996
Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs. Individual variation in the toxicity and therapeutic efficacy of these drugs is associated with a common genetic polymorphism that controls levels of TPMT activity and immunoreactive protein in human tissues. Because of the clinical significance of the "pharmacogenetic" regulation of this enzyme, it would be important to clone the gene for TPMT in humans and to study the molecular basis for the genetic polymorphism. As a first step toward cloning the gene for TPMT, we used the rapid amplification of genomic DNA ends to obtain a TPMr-specific intron sequence. That DNA sequence was used to design primers for the polymerase chain reaction (PCR), which made it possible to determine that the active gene for TPMT is located on human chromosome 6. A TPMr-positive cosmid clone was then isolated from a human chromosome 6-specific genomic DNA library, and the gene was sublocalized to chromosome band 6p22.3 by fluorescence in situ hybridization. The gene for TPMT was found to be approximately 34 kb in length and consisted of 10 exons and 9 introns. On the basis of the results of 5'-rapid amplification of cDNA ends, transcription initiation occurred at or near a point 89
Biochemical Pharmacology, 2010
Thiopurine S-methyltransferase (TPMT) genetic polymorphisms represent a striking example of the clinical importance of pharmacogenetics [1-3]. TPMT also represents a model system for the study of pharmacogenetic functional mechanisms-especially mechanisms involving nonsynonymous SNPs-polymorphisms that alter the encoded amino acid sequence [3]. We pointed out several years ago that one important way by which nonsynonymous SNPs influence function is by altering the quantity of the encoded protein [4]. TPMT has proved to be a useful system in which to study mechanisms responsible for that effect. TPMT*3A, the most common variant allele in Caucasians [5], a variant allele with two nonsynonymous SNPs [6], results in striking decreases in both TPMT protein and enzyme activity [7-11]. A series of studies over the past decade has demonstrated that this process involves proteasome-mediated degradation, with the involvement of molecular chaperones [9-11] as well as autophagy [8]. Those
Thiopurine S-methyltransferase pharmacogenetics: variant allele functional and comparative genomics
Pharmacogenetics and Genomics, 2005
The thiopurine S-methyltransferase (TPMT) genetic polymorphism is one of the most 'mature' examples in pharmacogenetics. That is true because of its importance clinically for the individualization of thiopurine drug therapy and also because TPMT has provided novel insights into molecular mechanisms responsible for the functional effects of common genetic polymorphisms. This review will summarize the development of our understanding of the role of inheritance in the regulation of TPMT as well as the clinical implications of that genetic regulation. It will also summarize recent studies in which TPMT pharmacogenetics has enhanced our understanding of molecular mechanisms by which common polymorphisms influence or alter function. TPMT pharmacogenetics highlights the potential clinical importance of the translation of pharmacogenetics from bench to bedside, the potential for basic pharmacogenetic research to provide insight into mechanisms by which genetic polymorphisms can alter function, and the challenges associated with the achievement of both of those goals.
Proceedings of The National Academy of Sciences, 2005
Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs. TPMT genetic polymorphisms represent a striking example of the potential clinical value of pharmacogenetics. Subjects homozygous for TPMT*3A, the most common variant allele for low activity, an allele that encodes a protein with two changes in amino acid sequence, are at greatly increased risk for life-threatening toxicity when treated with standard doses of thiopurines. These subjects have virtually undetectable levels of TPMT protein.
Biochemical Pharmacology, 2005
Human thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. TPMT is genetically polymorphic and is associated with large interindividual variations in thiopurine drug toxicity and therapeutic efficacy. During routine genotyping of patients with Crohn's disease, one novel missense mutation, 365A > C (TPMT*19, Lys 122 Thr), and a recently described missense mutation, 488G > A (TPMT*16, Arg 163 His), were identified in a Caucasian and a Moroccan patient, respectively. Using a heterologous yeast expression system, kinetic parameters (K m and V max ) of the two variants with respect to 6-thioguanine S-methylation were determined and compared with those obtained with the wild-type enzyme. The Lys 122 Thr exchange did not significantly decrease the intrinsic clearance value (V max /K m ) of the variant enzyme. In contrast, the Arg 163 His substitution significantly decreased the intrinsic clearance value by three-fold. The Arg 163 is located in a highly conserved region of the human TPMT protein and, as such, the Arg 163 His substitution is expected to result in a marked reduction of enzyme activity, as confirmed by the in vitro data. Phenotyping by measurement of red blood cell TPMT activity indicated that the patient heterozygous for the Lys 122 Thr mutation had normal TPMT activity, whereas the patient heterozygous for the Arg 163 His mutation was an intermediate methylator, which demonstrated a positive correlation between TPMT phenotyping and the in vitro data. The identification of a novel non-functional allele of the TPMT gene improves our knowledge of the genetic basis of interindividual variability in TPMT activity. These data further enhance the efficiency of genotyping methods to predict patients at risk of an inadequate response to thiopurine therapy. #
Pharmacogenomics of Thiopurine S-Methyltransferase: Clinical Applicability of Genetic Variants
Clinical Applications of Pharmacogenetics, 2012
Here, we provide an overview of the genetic variants of thiopurine S-methyltransferase (TPMT) gene that influence inter-individual dosing of thiopurine drugs, to highlight a tangible benefit of translating genomic knowledge into clinical practice. Particular single nucleotide polymorphisms (SNPs) in TPMT g e n e h a v e p r o v e n t o b e a p p l i c a b l e f o r optimising the dosage in pursuit of maximum efficacy and minimum adverse effects. Thus,
Thiopurine pharmacogenetics: clinical and molecular studies of thiopurine methyltransferase
Drug metabolism and disposition: the biological fate of chemicals, 2001
Thiopurine drugs are used to treat patients with neoplasia and autoimmune disease as well as transplant recipients. These agents are metabolized, in part, by S-methylation catalyzed by thiopurine methyltransferase (TPMT). The discovery nearly two decades ago that levels of TPMT activity in human tissues are controlled by a common genetic polymorphism led to one of the best examples of the potential importance of pharmacogenetics for clinical medicine. Specifically, it is now known that patients with inherited very low levels of TPMT activity are at greatly increased risk for thiopurine-induced toxicity such as myelosuppression when treated with standard doses of these drugs, while subjects with very high activity may be undertreated. Furthermore, recent reports indicate that TPMT may be the target for clinically significant drug interactions and that this common genetic polymorphism might be a risk factor for the occurrence of therapy-dependent secondary leukemia. In parallel with t...