In vitro characterization of four novel non-functional variants of the thiopurine S-methyltransferase (original) (raw)
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Thiopurine S-methyltransferase (TPMT) pharmacogenetics: variant allele functional genomics
Clinical Pharmacology & Therapeutics, 2004
TPMT genetic polymorphisms influence thiopurine drug toxicity and efficacy. We set out to perform functional genomic studies of 7 human TPMT variant alleles, TPMT*5: to *11:, that had not previously been studied by expression in mammalian cells. After expression in COS-1 cells, allozymes encoded by these alleles displayed from 2.3% (*5:) to 97.5% (*7:) of the level of TPMT activity–corrected for transfection efficiency–found with the wild type (WT) allele. TPMT*5:, *6: and *11: all displayed less than 50% of WT activity. Quantitative Western analysis showed that level of immunoreactive protein correlated closely with level of activity for all allozymes except TPMT*6:. However, substrate kinetic studies of all of the recombinant allozymes showed that *6: had significantly elevated apparent Km values for both cosubstrates for the reaction, 6-mercaptopurine and S-adenosyl-L-methionine, when compared with WT. These results indicate that some of the TPMT alleles that have been reported to be associated with clinical consequences do not appear to be functionally impaired after expression in mammalian cells.Clinical Pharmacology & Therapeutics (2004) 75, P19–P19; doi: 10.1016/j.clpt.2003.11.072
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. #
Proceedings of the National Academy of Sciences, 1997
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).
British Journal of Haematology, 2000
S-Methylation by thiopurine methyltransferase (TPMT) is an important route of metabolism for the thiopurine drugs. About one in 300 individuals are homozygous for a TPMT mutation associated with very low enzyme activity and severe myelosuppression if treated with standard doses of drug. To validate the use of molecular genetic techniques for the detection of TPMT deficiency, we have determined red blood cell TPMT activity in 240 adult blood donors and 55 normal children. Genotype was determined by restriction fragment length analysis of polymerase chain reaction products in a cohort of 79 of the blood donors and five cases of azathioprineinduced myelosupression, and this confirmed a close relationship between genotype and phenotype. In 17 of the 24 cases in which mutations were found, DNA was also available from remission bone marrow. In one of these cases, DNA from the remission marrow sample indicated the presence of a non-mutated allele that had not been seen in the blast DNA sample obtained at presentation. These results indicate that polymerase chain reaction-based assays give reliable and robust results for the detection of TPMT deficiency, but that caution should be exercised in relying exclusively on DNA obtained from lymphoblasts in childhood leukaemia.
Cardiovascular & hematological agents in medicinal chemistry, 2017
Thiopurine S-methyltransferase (TPMT) enzyme metabolizes thiopurine drugs which are widely used in various disciplines as well as in leukemias. Individual enzyme activity varies depending on the genetic polymorphisms of TPMT gene located at chromosome 6. Up to 14% of population is known to have a decreased enzyme activity, and if treated with standard doses of thiopurines, these individuals are at the high risk of severe adverse drug reactions (ADR) as myelosuppression, gastrointestinal intolerance, pancreatitis and hypersensitivity. However, TPMT-deficient patients can successfully be treated with decreased thiopurine doses if enzyme status is identified by a prior testing. TPMT status identification is a pioneering experience in an application of a pharmacogenetic testing in clinical settings. 4 TPMT (*2,*3A, *3B, *3C) alleles are known to account for 80-95% of a decreased enzyme activity, and therefore, identifying the presence of these alleles supported by phenotypic measurement...
Annals of Clinical Biochemistry: International Journal of Laboratory Medicine, 2006
Background: We have developed a new thiopurine S-methyltransferase (TPMT) phenotyping method that measures TPMT activity in whole blood. To evaluate this assay, we compared it with conventional TPMT phenotyping, which uses a red blood cell (RBC) lysate and genotyping for analysis of common TPMT mutations. Methods: Whole-blood and RBC lysates were prepared from 402 patients' samples received for routine analysis. The TPMT activity of lysates was determined using 6-thioguanine as substrate with high-performance liquid chromatographic (HPLC) analysis and fluorimetric detection. DNA was extracted from buffy coats using phenol-chloroform extraction. A multiplex amplification refractory mutation system (ARMS) strategy was used to screen for the common TPMT mutations TPMT*2 and TPMT*3 (TPMT*3A, TPMT*3C and TPMT*3D). Results: TPMT activities were higher in the whole-blood (mean TPMT activity 51 nmol 6-MTG/gHb/h) compared with the RBC lysate (37 nmol 6-MTG/gHb/h). Overall, concordance wi...
European Journal of Clinical Pharmacology, 2004
Objective: Thiopurine drugs are commonly used in pediatric patients for the treatment of acute leukemia, organ transplantation and inflammatory diseases. They are catabolized by the cytosolic thiopurine methyltransferase (TPMT), which is subject to a genetic polymorphism. In children, enzyme activities are immature at birth and developmental patterns vary widely from one enzyme to another. The present study was undertaken to evaluate erythrocyte TPMT activity and the correlation between genotype and phenotype in different age groups from birth to adolescence and adulthood. Methods: The study included 304 healthy adult blood donors, 147 children and 18 neonates (cord bloods). TPMT activity was measured by liquid chromatography, and genotype was determined using a polymerase chain reaction reverse dot-blot analysis identifying the predominant TPMT mutant alleles (TPMT*3A, TPMT*3B, TPMT*3C, TPMT*2). Results: There was no significant difference in TPMT activity between cord bloods (n=18) and children (n=147) (17.48±4.04 versus 18.62±4.14 respectively, P=0.424). However, TPMT was significantly lower in children than in adults (19.34±4.09) (P=0.033). In the whole population, there were 91.9% homozygous wild type, 7.9% heterozygous mutants and 0.2% homozygous mutants. The frequency of mutant alleles was 3.0% for TPMT*3A, 0.7% for TPMT*2 and 0.4% for TPMT*3C. Conclusion: No impact of child development on TPMT activity could be evidenced, suggesting that TPMT activity is already mature at birth. The difference between children and adults was low with reduced clinical impact expected. When individual TPMT activity was compared with genotype, there was an overlapping region where subjects (4.5%, 12 adults, 9 children) were either homozygous wild type or heterozygous, with a TPMT activity below the antimode value. This result highlighted the importance of measuring TPMT activity to detect all patients at risk of thiopurine toxicity.