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Characterization of a novel sequence variant, TPMT*28, in the human thiopurine methyltransferase gene
Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. (Medicin)ORCID iD: 0000-0002-2809-7591
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Oncology UHL. (Medicin)
Department of Gastroenterology, Lund University.
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2010 (English)In: Pharmacogenetics and genomics, ISSN 1744-6880, Vol. 20, no 11, 700-707 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The activity of the human enzyme thiopurine methyltransferase (TPMT) varies greatly between individuals because of genetic polymorphism. TPMT is involved in the detoxification and activation of thiopurines such as 6-mercaptopurine, 6-thioguanine, and azathioprine. These drugs are used in the treatment of acute lymphoblastic leukemia and inflammatory bowel disease. A total of 29 sequence variants have been identified so far in the TPMT gene. However, most of these variants are rare and not fully characterized. METHODS AND RESULTS: In this study, we describe the identification and characterization of a novel TPMT sequence variant, originally found in a Swedish man of Italian origin. Sequencing of the variable number tandem repeats region of the TPMT promoter and exons III-X revealed a T-to-C transition at nucleotide 611, causing an amino acid substitution from isoleucine to threonine at amino acid 204, positioned in an α-helix, approximately 16 Å from the active site. This new variant was found in the patient and in his son. Both had intermediate enzyme activity (8.1 U/ml packed red blood cells and 8.8 U/ml packed red blood cells, respectively) and neither carried other variants in the coding region of the gene. To be able to study this variant in more detail, the TPMT*28 variant was expressed in Escherichia coli, and an in-vitro characterization of the variant revealed that the protein was destabilized and showed a stronger tendency towards degradation at 37°C than the wild-type protein. The individuals carrying the TPMT*28 variant had less TPMT protein and lower TPMT activity in both red and white blood cells compared with a wild-type control. CONCLUSIONS: We present a detailed in-vivo and in-vitro characterization of a novel TPMT sequence variant (TPMT*28) causing decreased TPMT activity. Individuals carrying TPMT*28 might have an increased risk for developing severe side effects if treated with conventional doses of thiopurines.

Place, publisher, year, edition, pages
2010. Vol. 20, no 11, 700-707 p.
Keyword [en]
pharmacogenetics; protein stability; right-angle light scattering; single nucleotide polymorphism; thiopurine methyltransferase
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-60741DOI: 10.1097/FPC.0b013e3283402ee4PubMedID: 20881512OAI: diva2:358919
Available from: 2010-10-25 Created: 2010-10-25 Last updated: 2015-05-29
In thesis
1. Biophysical Characterization of Thiopurine S-Methyltransferase: A Key enzyme in the Effects of Thiopurine Drugs
Open this publication in new window or tab >>Biophysical Characterization of Thiopurine S-Methyltransferase: A Key enzyme in the Effects of Thiopurine Drugs
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the treatment of leukemia and inflammatory bowel disease, thiopurines are commonly used drugs. Thiopurine S-methyltransferase (TPMT) is one of the drug metabolizing enzymes responsible of counteracting the formation of TGNs that will be incorporated into the DNA and RNA synthesis and thus induce apoptosis. TPMT is a polymorphic enzyme and to date about 30 different sequence variants have been identified. Individuals who are to be treated with thiopurines are genotyped and/ or phenotyped at the time of diagnosis in order to individualize the treatment, with thiopurine dosage adjusted to the TPMT activity. In the treatment of acute lymphoblastic leukemia (ALL) high-dose methotrexate (MTX) is administered intravenously during the consolidation phase of the therapy and used in lower doses in the other phases of the ALL therapy. In blood samples from 53 children with ALL, we found decreased TPMT enzyme activity after 66 hours infusion of high-dose MTX. TPMT was recombinantly expressed, and the potential binding of MTX to TPMT was investigated by a fluorescence method. This showed that MTX bound to TPMT at relevant plasma concentrations observed in patient samples. At the time of leukemia diagnosis, TPMT activity was not correlated with the genotype for TPMT wild-types, which demonstrates the importance of using genotyping as a golden standard for determination of TPMT status in individuals with haematological malignancies. The low enzyme activity of TPMT*2 and TPMT*5 protein was evaluated by expressing these sequence variants in Escherichia coli (E.coli), and then characterizing them biophysically. Our results showed that TPMT*2 and TPMT*5 in the native state did not bind the extrinsic probe anilinonaphthalene sulfonate (ANS), which shows that the three-dimensional structure is already affected and restructured in that state. Based on these findings, we concluded that ANS can be used to probe the status of the active site. In another study we investigated the characteristics of TPMT*6 and TPMT*8 and found that the cofactor, S-adenosylmethionine (SAM) had a stabilizing effect on those sequence variants and on TPMT wild-type. Analysis of the structure of the TPMT protein by nuclear magnetic resonance (NMR) spectroscopy, enabled partial assignment of the backbone residue, of 64% of the TPMT sequence. Forty residues in TPMT exhibited millisecond dynamics but only 15 of those residues could be assigned, which emphasizes the difficulties involved in determining the three-dimensional structure of TPMT by NMR spectroscopy. In conclusion the present studies contribute to the understanding of the molecular characteristics of TPMT.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. 71 p.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1462
National Category
Natural Sciences
urn:nbn:se:liu:diva-80194 (URN)978-91-7519-851-4 (ISBN)
Public defence
2012-09-07, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 09:15 (Swedish)
Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2013-10-04Bibliographically approved

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Lindqvist Appell, MalinWennerstrand, PatriciaPeterson, CurtMårtensson, Lars-Göran
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