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  • 1.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Wennerstrand, Patricia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Peterson, Curt
    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.
    Hertervig, Erik
    Department of Gastroenterology, Lund University.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Characterization of a novel sequence variant, TPMT*28, in the human thiopurine methyltransferase gene2010In: Pharmacogenetics and genomics, ISSN 1744-6880, Vol. 20, no 11, p. 700-707Article in journal (Refereed)
    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.

  • 2.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology . Linköping University, Faculty of Health Sciences.
    Wennerstrand, Patricia
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Skoglund, Karin
    Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology . Linköping University, Faculty of Health Sciences.
    Lars-Göran, Mårtensson
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Hertervig, Erik
    Lund University Hospital, Sweden.
    Peterson, Curt
    Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Oncology UHL.
    Explaining TPMT genotype/phenotype discrepancy by identification of a novel sequence variant, TPMT*272009In: 13th International Symposium on Purine and Pyrimidine metabolism in man, 2009Conference paper (Refereed)
    Abstract [en]

    Thiopurine methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism of thiopurine drugs. Owing to polymorphisms in the TPMT gene (TPMT*2-*22), the enzyme activity varies interindividually. Patients with reduced TPMT activity may develop adverse reactions when treated with standard doses of thiopurines. This work focuses on a TPMT genotype/phenotype discrepancy found in a patient during routine testing. The patient displayed very low TPMT enzyme activity and she was genotyped by pyrosequencing as being heterozygous for the 460G>A and 719A>G polymorphisms (TPMT*3A). Complete sequencing in combination with haplotyping of the TPMT gene revealed a novel sequence variant, 500C>G, on one allele and TPMT*3A on the other allele, giving rise to the novel genotype TPMT*3A/*23. When investigating the patient's relatives, they too had the TPMT*3A/*23 genotype in combination with low enzyme activity. We conclude that this novel variant allele affects enzyme activity, as the individuals carrying it had almost undetectable TPMT activity.

  • 3.
    Wennerstrand, Patricia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    The Communication Guide2017Other (Other academic)
    Abstract [en]

    During the doctoral studies, it is essential that communication and expectations between PhD student and supervisor are clear and realistic.

    The need of supervision changes during the PhD period. It is therefore of importance that the PhD student and the supervisor take mutual responsibility in the communication and collaboration. The purpose of the communication guide is to stimulate the dialogue on these subjects.

  • 4.
    Wennerstrand, Patricia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Blissing, Annica Theresia
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    In vitro Protein Stability of Two Naturally Occurring Thiopurine S-methyltransferase Sequence Variants: Biophysical Characterization of TPMT*6 and TPMT*82017In: ACS Omega, E-ISSN 2470-1343, Vol. 2, no 8, p. 4991-4999Article in journal (Refereed)
    Abstract [en]

    Thiopurine S-methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism and inactivation of thiopurine substances administered as immunosuppressants in the treatment of malignancies and autoimmune diseases. In this study, the naturally occurring variants, TPMT*6 (Y180F) and TPMT*8 (R215H), have been biophysically characterized. Despite being classified as low and intermediate in vivo enzyme activity variants, respectively, our results demonstrate a discrepancy because both TPMT*6 and TPMT*8 were found to exhibit normal functionality in vitro. While TPMT*8 exhibited biophysical properties almost indistinguishable from those of TPMTwt, the TPMT*6 variant was found to be destabilized. Furthermore, the contributions of the cofactor S-adenosylmethionine (SAM) to the thermodynamic stability of TPMT were investigated, but only a modest stabilizing effect was observed. Also presented herein is a new method for studies of the biophysical characteristics of TPMT and its variants using the extrinsic fluorescent probe 8-anilinonaphthalene-1-sulfonic acid (ANS). ANS was found to bind strongly to all investigated TPMT variants with a Kd of approximately 0.2 μM and a 1:1 binding ratio as determined by isothermal titration calorimetry (ITC). Circular dichroism and fluorescence measurements showed that ANS binds exclusively to the native state of TPMT, and binding to the active site was confirmed by molecular modeling and simulated docking as well as ITC measurements. The strong binding of the probe to native TPMT and the conformity of the obtained results demonstrate the advantages of using ANS binding characteristics in studies of this protein and its variants.

  • 5.
    Wennerstrand, Patricia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Blissing, Annica Theresia
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Partially Assigned Chemical Shifts of Human Thiopurine S-methyltransferase Reveal Flexibility in Native StructureManuscript (preprint) (Other academic)
  • 6.
    Wennerstrand, Patricia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Dametto, Paolo
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Hennig, Janosch
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Klingstedt, Therése
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Skoglund, Karin
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Structural Characteristics Determine the Cause of the Low Enzyme Activity of Two Thiopurine S-Methyltransferase Allelic Variants: A Biophysical Characterization of TPMT*2 and TPMT*52012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 30, p. 5912-5920Article in journal (Refereed)
    Abstract [en]

    The enzyme thiopurine S-methyltransferase (TPMT) is involved in the metabolism of thiopurine drugs used to treat acute lymphoblastic leukemia and inflammatory bowel disease. Thus far, at least 29 variants of the TPMT gene have been described, many of which encode proteins that have low enzyme activity and in some cases become more prone to aggregation and degradation. Here, the two naturally occurring variants, TPMT*2 (Ala80 → Pro) and TPMT*5 (Leu49 → Ser), were cloned and expressed in Escherichia coli. Far-UV circular dichroism spectroscopy showed that TPMT*2 was substantially destabilized whereas TPMT*5 showed much greater stability comparable to that of wild-type TPMT (TPMTwt). The extrinsic fluorescent molecule anilinonaphthalene sulfonate (ANS) was used to probe the tertiary structure during thermal denaturation. In contrast to TPMTwt, neither of the variants bound ANS to a large extent. To explore the morphology of the TPMT aggregates, we performed luminescent conjugated oligothiophene staining and showed fibril formation for TPMT*2 and TPMT*5. The differences in the flexibility of TPMTwt, TPMT*2, and TPMT*5 were evaluated in a limited proteolysis experiment to pinpoint stable regions. Even though there is only one amino acid difference between the analyzed TPMT variants, a clear disparity in the cleavage patterns was observed. TPMT*2 displays a protected region in the C-terminus, which differs from TPMTwt, whereas the protected regions in TPMT*5 are located mainly in the N-terminus close to the active site. In conclusion, this in vitro study, conducted to probe structural changes during unfolding of TPMT*2 and TPMT*5, demonstrates that the various causes of the low enzyme activity in vivo could be explained on a molecular level.

  • 7.
    Wennerstrand, Patricia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Dametto, P
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Hennig, Janosch
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Skoglund, Karin
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Peterson, Curt
    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.
    Different mechanisms behind low enzyme activity in vivo of two different variants of Thiopurine S-methyltransferase, TPMT2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no Suppl. 1, p. 257-258Article in journal (Other academic)
    Abstract [en]

    In treatment of acute lymphoblastic leukemia and inflammatorybowel disease (IBD) thiopurines such as azathioprine and 6-mercaptopurineare used. All of these drugs are prodrugs and are, inthe cell, converted to 6-thioguanines (6-TGNs) and incorporatedinto DNA or inhibiting purine synthesis. A key enzyme for thisregulation is the cytosolic enzyme thiopurine S-methyltransferase(TPMT). This enzyme degrades azathioprine and 6-mercaptopurineto methylmercapto-purine and thereby reduces the bioavailabilityof the 6-TGNs incorporated into DNA. TPMT is apolymorphic enzyme with at least 29 different allelic variantsknown today and is one of the more classical examples of pharmacogeneticswhere the TPMT enzyme activity of the allelic variantsis directly correlated to the clinical dosages of the thiopurines, with a 10–15 fold dosage reduction for an allelic variantwith low TPMT enzyme activity. Even though TPMT is awell studied protein. Many studies have been performed in yeast‘‘suspensions’’ and not on pure protein solutions. It has beenspeculated and in a few cases shown that the reason for the lowactivity for most of the allelic variants is mainly due to the lowstability and/or tendency to aggregate. The mutations in thisstudy TPMT *2 (A80P) and TPMT * 5 (L49S) are both situatedat a distance far from the active site, however the enzyme activitiesare severely affected at 37°C. Preliminary results, using a repertoireof techniques such as CD, fluorescence and limitedproteolysis experiments suggest two different mechanisms for thelow enzyme activity at a temperature corresponding to in vivo conditions.

  • 8.
    Wennerstrand, Patricia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Söderhäll, Stefan
    Childhood Cancer Research Unit, Department of Women and Child Health, Astrid Lindgren Children's Hospital, Karolinska University Hospital, Stockholm.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Zimdahl, Anna
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia2013In: European Journal of Clinical Pharmacology, ISSN 0031-6970, E-ISSN 1432-1041, Vol. 69, no 9, p. 1641-1649Article in journal (Refereed)
    Abstract [en]

    Purpose

    Important drugs in the treatment of childhood acute lymphoblastic leukaemia (ALL) are 6-mercaptopurine (6-MP) and methotrexate (MTX). Thiopurine methyltransferase (TPMT) is a polymorphic enzyme causing variability in 6-MP response and toxicity. The aim of this study was to investigate the fluctuation in TPMT enzyme activity over time and the effect of high-dose MTX infusions on TPMT enzyme activity and 6-MP metabolites in paediatric ALL patients.

    Methods

    Fifty-three children with ALL treated according to the NOPHO-ALL 2000 protocol were included in the study. TPMT enzyme activity was measured at six different times starting from diagnosis until after the end of maintenance treatment. TPMT and 6-MP metabolites were measured before the initiation of high-dose MTX (HD-MTX) infusions and at 66 h post-infusion. The interaction between MTX and TPMT was investigated in vitro using recombinant TPMT protein and a leukaemic cell line.

    Results

    Forty percent of TPMT wild-type individuals had deceptively low TPMT enzyme activity according to genotype at the time of diagnosis. TPMT activity had decreased significantly 66 h after the start of HD-MTX infusions (−9.2 %; p = 0.013). MTX bound to recombinant TPMT protein severely inhibiting TPMT enzyme activity (remaining activity 16 %).

    Conclusions

    Our results show that TPMT genotyping should be performed in children with ALL, since 40 % of the children in our study who carried the wild-type TPMT gene were at risk of initial underdosing of 6-MP in cases where only TPMT enzyme activity was determined. MTX inhibits the TPMT enzyme activity after HD-MTX infusions due to protein binding.

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