Pharmacogenetics of TPMT: Screening for new biochemical factors influencing TPMT activity

  • Smid A
  • Milek M
  • Karas-Kuzelicki N
  • et al.
PMID: 70526934
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Abstract

Introduction: The main challenge of chemotherapy is to design individualized dosage regimens to overcome the boundaries of narrow therapeutic index. Both the efficacy and the toxicity of thiopurine drugs depend largely on the extent of their deactivation by S-methylation. This process is catalyzed by thiopurine S-methyltransferase (TPMT), a genetically polymorphic enzyme. Non-synonymous amino acid substitutions in mutant TPMT allozymes destabilize the protein structure and increase its susceptibility to proteasomal degradation. In homozygous mutants, TPMT activity is low or absent, while heterozygous individuals exhibit intermediate TPMT activity. In addition to the role played by genotype, TPMT activity is regulated by a complex metabolic network. As an SAM-dependant methyltransferase (MT-ase), TPMT is closely connected to the L-methionine (Met) cycle, the folate pathway, trans-sulfuration and glutathione (GSH) synthesis (Fig. 1). (Figure presented) Recently, some studies have reported on the post-translational stabilization of TPMT by its co-factor S-adenosylmethionine (SAM) (1) while others demonstrated that mutations in MTHFR, the enzyme involved in SAM biosythesis correlates well with hematotoxic events during ALL treatment, improving the predictive value of TPMT-based genotyping (2, 3). In this study we investigated the influence of SAM-related metabolites - namely GSH, B12, Hcy and folates- as well as of TPMT genotype and SAM levels, on TPMT activity in healthy individuals. Materials and Methods Study participants, preparation of hemolysates and genotyping assays. Blood samples were collected from 154 healthy individuals, donors of the Estonian Gene Bank. Genotyping of TPMT*3 alleles (460G>A - rs1800460, and 719A>G - rs1142345) was carried out by Taqman Genotyping Assays (Applied Biosystems, Foster City, CA) as described previously (2). Hemolysates were prepared in accordance with the previously described procedure (4, 5). An aliquot (0.1 ml) was used for the routine hemoglobin measurement on a Coulter Ac T Diff analyzer (Beckman Coulter, Brea, CA). Metabolites and TPMT activity. TPMT activity and SAM levels in hemolysates were determined using modified versions of the high-performance liquid chromatography (HPLC) methods described previously (1, 5). GSH levels were determined using the Glutathione Assay Kit from Sigma- Aldrich (St. Louis, Missouri, USA) in accordance with the manufacturer's instructions. B12, folate and Hcy levels were measured as part of routine blood analysis performed in the United Laboratories of Tartu University Hospital. Statistical analyses Correlations were determined by the linear regression model. Statistical analysis was carried out using PASW 18 (SPSS Inc., Chicago, IL, USA). RESULTS AND DISCUSSION Biochemical measurements, genotyping and determination of TPMT activity were performed in 154 healthy individuals. As expected, the results demonstrated significantly higher values of TPMT activity in individuals with wild type TPMT genotype (TPMT*1/1) compared to individuals with heterozygous TPMT genotype (TPMT*3/1) (Fig. 2). (Figure presented) Furthermore, the results showed that TPMT activity is influenced not only by TPMT genotype, but also by SAM levels (Fig. 3). (Figure presented) In the present set of samples, we could not observe statistically significant correlation between TPMT activity and B12, total folate levels and GSH levels. Conclusions Identifying and understanding the factors influencing TPMT activity are crucial for improving the efficacy and safety of thiopurine therapy. Our study demonstrated that TPMT activity is not influenced by TPMT genotype alone, but also by SAM levels and therefore indicates SAM as a potential novel biomarker in the individualization of thiopurine therapy.

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Smid, A., Milek, M., Karas-Kuzelicki, N., Tamm, R., Metspalu, A., & Mlinaric-Rascan, I. (2011). Pharmacogenetics of TPMT: Screening for new biochemical factors influencing TPMT activity. European Journal of Pharmaceutical Sciences, 1), 40–42. Retrieved from http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed12&AN=70526934 http://www.ovid.com/site/resources/expert_search/healthexp.html

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