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  • 1.
    Chaabane, Wiem
    et al.
    Linköping University, Faculty of Medicine and Health Sciences. Linköping University, Department of Medical and Health Sciences, Division of Drug Research.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Interconnections between apoptotic and autophagic pathways during thiopurine-induced toxicity in cancer cells: the role of reactive oxygen species2016In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 46, p. 75616-75634Article in journal (Refereed)
    Abstract [en]

    Thiopurines (azathioprine, 6-mercaptopurine and 6-thioguanine) are a class of genotoxic drugs extensively used in the treatment of various illnesses including leukemia. Their underlying molecular mechanism of action involves the activation of apoptosis and autophagy but remains widely unclear. Here we present evidence that autophagy induction by thiopurines is a survival mechanism that antagonizes apoptosis and is involved in degrading damaged mitochondria through mitophagy. On the other hand, apoptosis is the main cell death mechanism by thiopurines as its inhibition prohibited cell death. Thus a tight interplay between apoptosis and autophagy controls cell fate in response to thiopurine treatment. Moreover, thiopurines disrupt mitochondrial function and induce a loss of the mitochondrial transmembrane potential. The involvement of the mitochondrial pathway in thiopurine-induced apoptosis was further confirmed by increased formation of reactive oxygen species (ROS). Inhibiting oxidative stress protected the cells from thiopurine-induced cell death and ROS scavenging prohibited autophagy induction by thiopurines. Our data indicate that the anticarcinogenic effects of thiopurines are mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis and autophagy.

  • 2.
    Coulthard, Sally A
    et al.
    Newcastle University.
    Redfern, Christopher P F
    Linköping University, Department of Medical and Health Sciences, 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.
    Skoglund, Karin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Jakobsen Falk, Ingrid
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Hall, Andrew G
    Newcastle University.
    Taylor, Gordon A
    Newcastle University.
    Hogarth, Linda A
    Newcastle University.
    Increased Sensitivity to Thiopurines in Methylthioadenosine Phosphorylase-Deleted Cancers2011In: MOLECULAR CANCER THERAPEUTICS, ISSN 1535-7163, Vol. 10, no 3, p. 495-504Article in journal (Refereed)
    Abstract [en]

    The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), are used in the treatment of leukemia. Incorporation of deoxythioguanosine nucleotides (dG(s)) into the DNA of thiopurine-treated cells causes cell death, but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS). The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Because the growth of MTAP-deleted tumor cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the T-cell acute lymphoblastic leukemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP(-)) transfected to express MTAP constitutively (A549-MTAP(+)). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intracellularly to MeTIMP, was markedly higher in both cell lines under MTAP(-) conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dG(s) incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP.

  • 3.
    Coulthard, Sally
    et al.
    Newcastle University, UK.
    Redfern, Christopher
    Newcastle University, UK.
    Vikingsson, Svante
    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.
    Skoglund, Karin
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Jakobsen Falk, Ingrid
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Hall, Andrew
    Newcastle University, UK.
    Taylor, Gordon
    Newcastle University, UK.
    Hogarth, Linda
    Newcastle University, UK.
    Increased sensitivity to thiopurines in methylthioadenosine phosphorylase-deleted cancers in PURINERGIC SIGNALLING, vol 6, issue , pp 33-332010In: PURINERGIC SIGNALLING, Springer Science Business Media , 2010, Vol. 6, p. 33-33Conference paper (Refereed)
    Abstract [en]

    The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used in the treatment of leukaemia. Incorporation of deoxythioguanosine nucleotides (dGs) into the DNA of thiopurine-treated cells causes cell death but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS). The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Since the growth of MTAP-deleted tumour cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the T-cell acute lymphoblastic leukaemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP-ve) transfected to express MTAP constitutively (A549-MTAP+ve). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intra-cellularly to MeTIMP, was markedly higher in both cell lines under MTAP-ve conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dGs incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP.

  • 4.
    Fotoohi, Alan Kambiz
    et al.
    KI, Stockolm.
    Lindqvist Appell, Malin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Albertioni, Freidoun
    KI, Stockholm.
    Impaired transport as a mechanism of resistance to thiopurines in human T-lymphoblastic leukemia cells2006In: Nucleosides, Nucleotides & Nucleic Acids, ISSN 1525-7770, E-ISSN 1532-2335, Vol. 25, no 9-11, p. 1039-1044Article in journal (Refereed)
    Abstract [en]

    In order to better understand the mechanisms of resistance to thiopurines, we studied two sublines of the MOLT4 T-lymphoblastic leukemia cell line, resistant to 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). We found that the underlying mechanism of resistance in both resistant cell lines was a markedly reduction in initial transport of 6-MP (3- and 5-fold, respectively, in 6-MP- and 6-TG-resistant cells). No significant alteration of activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase, the key enzymes involved in the metabolism of thiopurines was detected. We conclude that defected initial transport of thiopurines by cells may very well explain their resistance to these drugs. Copyright © Taylor & Francis Group, LLC.

  • 5.
    Fotoohi, Alan Kambiz
    et al.
    KI, Stockholm.
    Lindqvist Appell, Malin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Albertioni, Freidoun
    KI, Stockholm.
    Involvement of the concentrative nucleoside transporter 3 and equilibrative nucleoside transporter 2 in the resistance of T-lymphoblastic cell lines to thiopurines2006In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 343, no 1, p. 208-215Article in journal (Refereed)
    Abstract [en]

    Mechanisms of resistance to thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) were investigated in human leukemia cell lines. We developed two 6-MP- and 6-TG-resistant cell lines from the human T-lymphoblastic cell line (MOLT-4) by prolonged exposure to these drugs. The resistant cells were highly cross resistant to 6-MP and 6-TG, and exhibited marked reduction in cellular uptake of 6-MP (70% and 80%, respectively). No significant modification of the activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase was observed. Real-time PCR of concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2) of resistant cells showed substantial reductions in expression of messenger RNAs. Small interfering RNA designed to silence the CNT3 and ENT2 genes down-regulated the expression of these genes in leukemia cells. These decreases were accompanied by reduction of transport of 6-MP (47% and 21%, respectively) as well as its cytocidal effect (30% and 21%, respectively). Taken together these results show that CNT3 and ENT2 play a key role in the transport of 6-MP and 6-TG by leukemia cells. From a clinical point of view determination of CNT3 and ENT2 levels in leukemia cells may be useful in predicting the efficacy of thiopurine treatment. © 2006 Elsevier Inc. All rights reserved.

  • 6.
    Fransson, Martin
    et al.
    Linköping University, Department of Computer and Information Science, PELAB - Programming Environment Laboratory. Linköping University, The Institute of Technology.
    Fritzson, Peter
    Linköping University, Department of Computer and Information Science, PELAB - Programming Environment Laboratory. Linköping University, The Institute of Technology.
    Lindqvist Appell, Malin
    Linköping University, Department of Medicine and Care. Linköping University, Faculty of Health Sciences.
    Hindorf, Ulf
    Almer, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    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.
    A preliminary study of modeling and simulation in individualized drug dosage – azathioprine on inflammatory bowel disease2007In: SIMS 2006: Proceedings of the 47th Conference on Simulation and Modelling, Helsinki, Finland, Helsinki: Kopio Niini Oy , 2007, p. 216-220Conference paper (Refereed)
    Abstract [en]

    Individualized drug dosage based on population pharmacokinetic/dynamic models is an important future technology used to reduce or eliminate side effects of certain drugs, e.g. cancer drugs. In this paper we report preliminary results from work-in-progress: a simplified linear model of the metabolism of a cancer treatment drug was estimated from experimental data. The model was then validated against the same data as a test of the adequacy of the model structure. From this investigation it became apparent that the model structure could not be used due to its inability to recreate the dynamic properties of the system.

  • 7.
    Gerbek, Tina
    et al.
    Univ Hosp Copenhagen, Denmark.
    Ebbesen, Maria
    Univ Hosp Copenhagen, Denmark.
    Nersting, Jacob
    Univ Hosp Copenhagen, Denmark.
    Frandsen, Thomas L.
    Univ Hosp Copenhagen, Denmark.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Schmiegelow, Kjeld
    Univ Copenhagen, Denmark.
    Role of TPMT and ITPA variants in mercaptopurine disposition2018In: Cancer Chemotherapy and Pharmacology, ISSN 0344-5704, E-ISSN 1432-0843, Vol. 81, no 3, p. 579-586Article in journal (Refereed)
    Abstract [en]

    To explore the levels of thioguanine incorporated into DNA (DNA-TG), and erythrocyte levels of 6-thioguanine nucleotides (Ery-TGN) and methylated metabolites (Ery-MeMP) during 6-mercaptopurine (6MP)/Methotrexate (MTX) therapy of childhood acute lymphoblastic leukemia (ALL) and the relation to inosine triphosphatase (ITPA) and thiopurine methyltransferase (TPMT) gene variants. Blood samples were drawn during 6MP/MTX maintenance therapy from 132 children treated for ALL at Rigshospitalet, Copenhagen. The samples were analysed for thiopurine metabolites and compared to TPMT (rs1800460 and rs1142345) and ITPA (rs1127354) genotypes. Median DNA-TG (mDNA-TG) levels were higher in TPMT and ITPA low-activity patients as compared to wildtype patients (TPMT (LA) 549 vs. 364 fmol/A mu g DNA, p = 0.007, ITPA (LA) 465 vs. 387 fmol/A mu g DNA, p = 0.04). mDNA-TG levels were positively correlated to median Ery-TGN (mEry-TGN)(r (s) = 0.37, p = 0.001), but plateaued at higher mEry-TGN levels. DNA-TG indices (mDNA-TG/mEry-TGN) were 42% higher in TPMT (WT) patients as compared to TPMT (LA) patients but no difference in DNA-TG indices was observed between ITPA (WT) and ITPA (LA) patients (median 1.7 vs. 1.6 fmol/A mu g DNA/ nmol/mmol Hb, p = 0.81). DNA-TG indices increased with median Ery-MeMP (mEry-MeMP) levels (r (s) = 0.25, p = 0.001). TPMT and ITPA genotypes significantly influence the metabolism of 6MP. DNA-TG may prove to be a more relevant pharmacokinetic parameter for monitoring 6MP treatment intensity than cytosolic metabolites. Prospective trials are needed to evaluate the usefulness of DNA-TGN for individual dose adjustments in childhood ALL maintenance therapy.

  • 8.
    Green, Henrik
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pharmacology.
    Genetiska test för optimal dosering på väg att bli klinisk rutin2013In: Onkologi i Sverige, no 3, p. 36-40Article in journal (Other (popular science, discussion, etc.))
  • 9.
    Gréen, Henrik
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Lindqvist Appell, Malin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Zackrisson, Anna Lena
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Juliusson, Gunnar
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Hematology. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Haematology UHL.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    P-gp and mdr-1 mRNA in leukemic cells fromAML patients during chemotheraphy.2001In: Proceedings of the American Association for Cancer Research,2001, 2001, p. 345-355Conference paper (Refereed)
  • 10.
    Haglund, Sofie
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology . Linköping University, Faculty of Health Sciences.
    Lindqvist Appell, Malin
    Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology . Linköping University, Faculty of Health Sciences.
    Almer, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology . Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology . Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Oncology UHL.
    Taipalensuu, J.
    Div. of R. and D. in Lab. Medicine, Ryhov County Hospital, SE-551 85 Jönköping, Sweden.
    Pyrosequencing of TPMT Alleles in a General Swedish Population and in Patients with Inflammatory Bowel Disease2004In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 50, no 2, p. 288-295Article in journal (Refereed)
    Abstract [en]

    Background: Interindividual differences in therapeutic efficacy in patients treated with thiopurines might be explained by the presence of thiopurine S-methyltransferase (TPMT) alleles that encode for reduced TPMT enzymatic activity. It is therefore of value to know an individual's inherent capacity to express TPMT. Method: We developed a pyrosequencing method to detect 10 single-nucleotide polymorphisms (SNPs) in TPMT. A Swedish population (n = 800) was examined for TPMT*3A, TPMT*3B, TPMT*3C, and TPMT*2. Patients with inflammatory bowel disease (n = 24) and healthy volunteers (n = 6), selected on the basis of TPMT enzymatic activity, were investigated for all 10 SNPs to determine the relationship between TPMT genotype and phenotype. Results: In the general population we identified the following genotypes with nonfunctional alleles: TPMT*1/*3A (*3A allelic frequency, 3.75%), TPMT*1/*3C (*3C allelic frequency, 0.44%), TPMT*1/*3B (*3B allelic frequency, 0.13%), and TPMT*1/*2 (*2 allelic frequency, 0.06%). All nine individuals with normal enzymatic activity were wild-type TPMT*1/*1. Thirteen individuals with intermediate activity were either TPMT*1/*3A (n = 12) or TPMT*1/*2 (n = 1). Eight individuals with low enzymatic activity were TPMT*3A/*3A (n = 4), TPMT*3A/*3C (n = 2), or TPMT*1/*3A (n = 2). Conclusion: Next to wild type, the most frequent alleles in Sweden are TPMT*3A and TPMT*3C. A previously established phenotypic cutoff for distinguishing normal from intermediate metabolizers was confirmed. To identify the majority of cases (90%) with low or intermediate TPMT activity, it was sufficient to analyze individuals for only 3 of the 10 SNPs investigated. Nevertheless, this investigation indicates that other mutations might be of relevance for decreased enzymatic activity. © 2004 American Association for Clinical Chemistry.

  • 11.
    Hindorf, Ulf
    et al.
    Department of Gastroenterology, Skåne University Hospital, Lund, Sweden.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Genotyping should be considered the primary choice for pre-treatment evaluation of thiopurine methyltransferase function2012In: Journal of Crohn's and Colitis, ISSN 1873-9946, Vol. 6, no 6, p. 655-659Article in journal (Refereed)
    Abstract [en]

    Background and aims

    A pre-treatment determination of the thiopurine S-methyltransferase (TPMT) genotype or phenotype can identify patients at risk of developing severe adverse reactions from thiopurine treatment. The risk of misclassifying a patient might be dependent on the method used. The aim of this study was to investigate the concordance between TPMT genotyping and phenotyping.

    Methods

    The data consist of 7195 unselected and consecutive TPMT genotype and phenotype determinations sent to the division of Clinical Pharmacology, Linköping, Sweden. TPMT activity was measured in red blood cells (RBC) and the genotype determined by pyrosequencing for the three most common TPMT variants (TPMT *2, *3A, *3C).

    Results

    TPMT genotyping identified 89% as TPMT wild type (*1/*1), 10% as TPMT heterozygous and 0.5% as TMPT defective. The overall concordance between genotyping and phenotyping was 95%, while it was 96% among IBD patients (n = 4024). Genotyping would have misclassified 8% of the TPMT defectives as heterozygous as compared to 11% if only TPMT activity had been measured. 11% of the heterozygous patients had a normal TPMT activity (> 8.9 U/ml RBC) and 3% of the TPMT wild-type patients had an intermediate TPMT activity (2.5–8.9 U/ml RBC).

    Conclusions

    There is a risk for TPMT misclassification when only genotyping or phenotyping is used, but it is not reasonable to check both in all patients. Since TPMT genotyping is the more reliable test, especially in TPMT heterozygotes, we suggest that genotyping should be considered the primary choice for the pre-treatment evaluation of TPMT function before initiation of thiopurine therapy.

  • 12.
    Hindorf, Ulf
    et al.
    Skåne University Hospital, Lund, Sweden.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Letter: TPMT status determination: The simplest is the most effective? Reply: Reply to Dr. Chouchana's letter2012In: Journal of Crohn's & Colitis, ISSN 1873-9946, E-ISSN 1876-4479, Vol. 6, no 7, p. 808-808Article in journal (Other academic)
  • 13.
    Hindorf, Ulf
    et al.
    Lund.
    Lindqvist Appell, Malin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Hildebrand, Hans
    Stockholm.
    Fagerberg, Ulrika
    Stockolm.
    Almer, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Adverse events leading to modification of therapy in a large cohort of patients with inflammatory bowel disease2006In: Alimentary Pharmacology and Therapeutics, ISSN 0269-2813, E-ISSN 1365-2036, Vol. 24, no 2, p. 331-342Article in journal (Refereed)
    Abstract [en]

    Background: Adverse events leading to discontinuation or dose reduction of thiopurine therapy occur in 9-28% of patients with inflammatory bowel disease. Aims: To evaluate the influence of thiopurine methyltransferase status and thiopurine metabolites in a large patient population for the risk of developing adverse event. Methods: Three hundred and sixty-four patients with inflammatory bowel disease and present or previous thiopurine therapy were identified from a local database. Results: The adverse event observed in 124 patients (34%) were more common in adults than children (40% vs. 15%, P < 0.001) and in low to intermediate (≤9.0 U/mL red blood cell) than normal thiopurine methyltransferase activity (P = 0.02). Myelotoxicity developed later than other types of adverse event. An increased frequency of adverse event was observed in patients with tioguanine (thioguanine) nucleotide above 400 or methylated thioinosine monophosphate above 11 450 pmol/ 8 × 108 red blood cell. A shift to mercaptopurine was successful in 48% of azathioprine-intolerant patients and in all cases of azathioprine-induced myalgia or arthralgia. Conclusions: A pre-treatment determination of thiopurine methyltransferase status might be appropriate as patients with low to intermediate thiopurine methyltransferase activity are more prone to develop an adverse event, determination of metabolite levels can be useful in the case of an adverse event. Mercaptopurine therapy should be considered in azathioprine-intolerant patients. © 2006 The Authors.

  • 14.
    Hindorf, Ulf
    et al.
    Lund .
    Lindqvist Appell, Malin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Söderkvist, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Ström, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Hjortswang, Henrik
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Pousette, A
    Almer, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Pharmacogenetics during standardised initiation of thiopurine treatment in inflammatory bowel disease2006In: Gut, ISSN 0017-5749, E-ISSN 1468-3288, Vol. 55, no 10, p. 1423-1431Article in journal (Refereed)
    Abstract [en]

    Background: Firm recommendations about the way thiopurine drugs are introduced and the use of thiopurine methyltransferase (TPMT) and metabolite measurements during treatment in inflammatory bowel disease (IBD) are lacking. Aim: To evaluate pharmacokinetics and tolerance after initiation of thiopurine treatment with a fixed dosing schedule in patients with IBD. Patients: 60 consecutive patients with Crohn's disease (n = 33) or ulcerative colitis (n = 27) were included in a 20 week open, prospective study. Methods: Thiopurine treatment was introduced using a predefined dose escalation schedule, reaching a daily target dose at week 3 of 2.5 mg azathioprine or 1.25 mg 6-mercaptopurine per kg body weight. TPMT and ITPA genotypes, TPMT activity, TPMT gene expression, and thiopurine metabolites were determined. Clinical outcome and occurrence of adverse events were monitored. Results: 27 patients completed the study per protocol, while 33 were withdrawn (early protocol violation (n = 5), TPMT deficiency (n = 1), thiopurine related adverse events (n = 27)), 67% of patients with adverse events tolerated long term treatment on a lower dose (median 1.32 mg azathioprine/kg body weight). TPMT activity did not change during the 20 week course of the study but a significant decrease in TPMT gene expression was found (TPMT/huCYC ratio, p = 0.02). Patients with meTIMP concentrations > 11 450 pmol/8 × 108 red blood cells during steady state at week 5 had an increased risk of developing myelotoxicity (odds ratio = 45.0, p = 0.015). Conclusions: After initiation of thiopurine treatment using a fixed dosing schedule, no general induction of TPMT enzyme activity occurred, though TPMT gene expression decreased. The development of different types of toxicity was unpredictable, but we found that measurement of meTIMP early in the steady state phase helped to identify patients at risk of developing myelotoxicity.

  • 15.
    Hindorf, Ulf
    et al.
    Lunds Universitet.
    Lindqvist Appell, Malin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Söderkvist, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Ström, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Hjortswang, Henrik
    Linköping University, Department of Molecular and Clinical Medicine.
    Pousette, Anneli
    Almer, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    High methylthioinosine monophosphate levels as a cause of myelotoxicity when introducing thiopurine therapy in patients with inflammatory bowel disease2005In: 13th United European Gastroenterology week,2005, Stuttgart: Georg Thieme Verlag KG , 2005, p. A169-Conference paper (Refereed)
  • 16.
    Kalman, L. V.
    et al.
    Centre Disease Control and Prevent, GA USA.
    Agundez, J. A. G.
    University of Extremadura, Spain.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Black, J. L.
    Mayo Clin, MN USA.
    Bell, G. C.
    University of S Florida, FL 33682 USA.
    Boukouvala, S.
    Democritus University of Thrace, Greece.
    Bruckner, C.
    Affymetrix, CA USA.
    Bruford, E.
    European Molecular Biol Lab, England.
    Caudle, K.
    St Jude Childrens Research Hospital, TN 38105 USA.
    Coulthard, S. A.
    Newcastle University, England.
    Daly, A. K.
    Newcastle University, England.
    Del Tredici, A. L.
    Millennium Health LLC, CA USA.
    den Dunnen, J. T.
    Leiden University, Netherlands.
    Drozda, K.
    US FDA, MD USA.
    Everts, R. E.
    Agena Bioscience, CA USA.
    Flockhart, D.
    Indiana University of School Med, IN 46202 USA.
    Freimuth, R. R.
    Mayo Clin, MN USA.
    Gaedigk, A.
    University of Missouri, MO 64110 USA; University of Missouri, MO 64108 USA.
    Hachad, H.
    Translat Software, WA USA.
    Hartshorne, T.
    Thermo Fisher Science, CA USA.
    Ingelman-Sundberg, M.
    Karolinska Institute, Sweden.
    Klein, T. E.
    Stanford University, CA 94305 USA.
    Lauschke, V. M.
    Karolinska Institute, Sweden.
    Maglott, D. R.
    National Lib Med, MD USA.
    McLeod, H. L.
    University of S Florida, FL 33682 USA.
    McMillin, G. A.
    University of Utah, UT USA; ARUP Labs, UT USA.
    Meyer, U. A.
    University of Basel, Switzerland.
    Mueller, D. J.
    University of Toronto, Canada.
    Nickerson, D. A.
    University of Washington, WA 98195 USA.
    Oetting, W. S.
    University of Minnesota, MN USA.
    Pacanowski, M.
    US FDA, MD USA.
    Pratt, V. M.
    Indiana University of School Med, IN 46202 USA.
    Relling, M. V.
    St Jude Childrens Research Hospital, TN 38105 USA.
    Roberts, A.
    Aegis Science Corp, TN USA.
    Rubinstein, W. S.
    National Lib Med, MD USA.
    Sangkuhl, K.
    Stanford University, CA 94305 USA.
    Schwab, M.
    Dr Margarete Fischer Bosch Institute Clin Pharmacol, Germany; University Hospital, Germany.
    Scott, S. A.
    Icahn School Medical Mt Sinai, NY 10029 USA.
    Sim, S. C.
    Karolinska Institute, Sweden.
    Thirumaran, R. K.
    Genelex Corp, WA USA.
    Toji, L. H.
    Coriell Institute Medical Research, NJ USA.
    Tyndale, R. F.
    University of Toronto, Canada.
    van Schaik, R. H. N.
    Erasmus MC, Netherlands.
    Whirl-Carrillo, M.
    Stanford University, CA 94305 USA.
    Yeo, K. T. J.
    University of Chicago, IL 60637 USA.
    Zanger, U. M.
    Dr Margarete Fischer Bosch Institute Clin Pharmacol, Germany; University Hospital, Germany.
    Pharmacogenetic allele nomenclature: International workgroup recommendations for test result reporting2016In: Clinical Pharmacology and Therapeutics, ISSN 0009-9236, E-ISSN 1532-6535, Vol. 99, no 2, p. 172-185Article in journal (Refereed)
    Abstract [en]

    This article provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward.

  • 17.
    Karim, Hazhar
    et al.
    Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Fotoohi, Alan
    Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.
    Comparison of three methods for measuring thiopurine methyltransferase activity in red blood cells and human leukemia cells2013In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 939, p. 80-85Article in journal (Refereed)
    Abstract [en]

    Thiopurine efficacy is partly reflected by the genetic polymorphism of the thiopurine methyltransferase (TPMT) enzyme, which is responsible for variation in the metabolism, toxicity and therapeutic efficacy of the thiopurines azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Determination of TPMT activity before administration of thiopurines is thus crucial for individualized dosing in order to prevent toxicity in TPMT deficient individuals. These individuals must be treated with markedly lower (eg, 5-10% of the standard) doses of the prescribed medications. This paper describes a comparison of three different methods for the quantification of TPMT activity in red blood cells (RBC) and cultured human cell lines. We succeeded to perform the measurement of TPMT activity in a minimum amount of 1×10(6) cultured cells with an HPLC-UV system modified and optimized in our laboratory. The TPMT activity was linearly correlated with the cell concentration of the cultured cell line in a range of 1-10×10(6) cells. A significant correlation of determination of TPMT activity in RBC between radiometric detection by HPLC, classic radiochemical detection and UV detection by HPLC, was observed, correlation coefficient (r) were 0.72 and 0.73, respectively. The within-day and day-to-day coefficients of variation of the HPLC-UV-based method were 8% and 16%, respectively. The evaluation of the methods was demonstrated by studying the TPMT activity in RBC isolated from 198 patients, as well as in MOLT4 leukemic cell line and its sub-cell lines with acquired resistance to 6-MP and 6-TG.

  • 18.
    Levinsen, Mette
    et al.
    University Hospital Rigshospitalet, Copenhagen, Denmark.
    Ørskov Rotevatn, Elisabeth
    University Hospital Rigshospitalet, Copenhagen, Denmark.
    Rosthøj, Susanne
    University of Copenhagen, Denmark.
    Nersting, Jacob
    University of Copenhagen, Denmark .
    Abrahamsson, Jonas
    Queen Sylvias Children Hospital, Gothenburg, Sweden.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Bergan, Stein
    Oslo University Hospital, Norway .
    Bechensteen, Anne-Grete
    Oslo University Hospital, Norway .
    Harila-Saari, Arja
    University Hospital, Oulu, Finland.
    Heyman, Mats
    Astrid Lindgrens Hospital, Stockholm, Sweden.
    Gisli Jonsson, Olafur
    University Hospital, Reykjavik, Iceland.
    Maxild, Jakob Bernhard Cohn
    University of Copenhagen, Denmark .
    Niemi, Mikko
    University of Helsinki, Finland.
    Söderhäll, Stefan
    Astrid Lindgrens Hospital, Stockholm, Sweden .
    Schmiegelow, Kjeld
    University Hospital Rigshospitalet, Copenhagen, Denmark.
    Pharmacogenetically Based Dosing of Thiopurines in Childhood Acute Lymphoblastic Leukemia: Influence on Cure Rates and Risk of Second Cancer2014In: Pediatric Blood & Cancer, ISSN 1545-5009, E-ISSN 1545-5017, Vol. 61, no 5, p. 797-802Article in journal (Refereed)
    Abstract [en]

    Background

    Previous studies have indicated that patients with thiopurine methyltransferase (TPMT) low activity (TPMTLA) have reduced risk of relapse but increased risk of second malignant neoplasm (SMN) compared to patients with TPMT wild-type (TPMTWT) when treated with 6MP maintenance therapy starting doses of 75 mg/m2/day. To reduce SMN risk, 6MP starting doses were reduced to 50 mg/m2/day for patients with TPMT heterozygosity in the Nordic Society of Paediatric Haematology and Oncology (NOPHO) ALL2000 protocol.

    Procedure

    We explored the pattern of SMN and relapse in the NOPHO ALL2000 protocol (n = 674) and NOPHO ALL92 protocol (n = 601) in relation to TPMT pheno- and/or genotype.

    Results

    The overall risk of any event did not differ significantly between the two protocols. However, in event pattern analyses considering only the patients with TPMTLA who experienced relapse or SMN, the risk of SMN versus leukemia relapse was significantly lower in the ALL2000 cohort for patients with a 6MP starting dose <75 mg/m2/day when compared to the patients in ALL92 (relapse (n = 11) and SMN (n = 0) in ALL2000 versus relapse (n = 5) and SMN (n = 4) in ALL92, P = 0.03). Furthermore, the 8-year cumulative incidence of relapse for patients with TPMTLA was significantly higher in the ALL2000 compared to the ALL92 cohort (19.7% (11.6–33.3%) vs. 6.7% (2.9–15.5%), P = 0.03).

    Conclusion

    This study indicates that reducing 6MP starting dose for patients with TPMTLA may reduce SMN risk but lead to a relapse risk similar to that of patients with TPMTWT.

  • 19.
    Lindqvist Appell, Malin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Pharmacogenetic studies of thiopurines - focus on thiopurine methyltransferase2005In: Gastrokuriren, ISSN 1651-0453, Vol. 10Article in journal (Other (popular science, discussion, etc.))
  • 20.
    Lindqvist Appell, Malin
    Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Pharmacogenetic studies of thiopurines: focus on thiopurine methyltransferase2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Pharmacogenetics represents the study ofvariability in drug response due to genetic variations. The thiopurines (6-mercaptopurine, 6-thioguanine and azathioprine) are prodrugs which require metabolic transformation to exert effect. Thiopurines are used in inflammatory bowel disease, as maintenance treatment of childhood acute lymphoblastic leukaemia, and for immunosuppression after transplantation. The metabolism is complex and one important enzyme involved is thiopurine methyltransferase (TPMT). Inherited variation in TPMT activity is one factor responsible for individual differences in susceptibility to thiopurine-induced toxicity or in the therapeutic response to thiopurines. The enzyme activity is under controi of agenetic polymorphism. The frequency distribution of TPMT activity in Caucasians is trimodal, with 89% having high enzyme activity, 10% having intermediate activity and l of 300 having almost undetectable activity. The TPMT gene has been characterised and several single nucleotide polymorphisms (SNPs) identified causing decreased enzyme activity. The most common SNPs are TPMT*2, TPMT*3A and TPMT*3C.

    In the investigations for this thesis we have studied the pharmacogenetics of thiopurines with focus on TPMT. A real-time RT-PCR method was developed for quantification of TPMT gene expression, and a pyrosequencing method was developed for genotyping of TPMT SNPs. TPMT gene expression correlated to enzyme activity in individuals with high enzyme activity. The allele frequencies of TPMT*3A and TPMT*3C in samples from 800 Swedish individuals were in agreement with those in other Caucasian populations, although TPMT*3B was more common and TPMT*2 was rarer.

    We investigated the concordance between genotype and phenotype and found discordance between genotype and phenotype in two unrelated patients. In these patients, we detected two new sequence variants, TPMT*14 and TPMT*15, which lead to a non-functional TPMT enzyme. The TPMT genotype and phenotype were also determined in the parents of the two patients and the inheritance of these alleles was investigated.

    Sixty patients with inflammatory bowel disease following a standardised dose escalation schedule of azathioprine or 6-mercaptopurine were closely monitored over the course of 20 weeks. During treatment, the TPMT gene expression decreased. In contrast, TPMT enzyme activity did not change. TPMT heterozygous patients had a lower probability of remaining in the 20week study. Forty-five percent of the patients were withdrawn due to adverse events, but 67% of these tolerated a lower dose of thiopurines. The inosine triphosphate pyrophosphatase polymorphism (ITPA 94C>A) was not associated with occurrences of adverse events.

  • 21.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Almer, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, MC - Medicincentrum, EMT-magtarm.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Söderkvist, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Real-time RT-PCR methodology for quantification of thiopurine methyltransferase gene expression2003In: European Journal of Clinical Pharmacology, ISSN 0031-6970, E-ISSN 1432-1041, Vol. 59, no 3, p. 207-211Article in journal (Refereed)
    Abstract [en]

    Objective: The aim of the present study was to develop a real-time reverse-transcription polymerase chain reaction (RT-PCR) methodology for the quantification of thiopurine methyltransferase (TPMT) gene expression in whole blood and compare it with the TPMT enzyme activity measured in red blood cells. Methods: TPMT gene expression was quantified relative to the housekeeping gene cyclophilin (huCYC) and expressed as a TPMT/huCYC ratio. TPMT activity in red blood cells was determined by measuring the formation rate of 6- 14C-methylmercaptopurine from 6-MP using S-adenosyl-L-( 14C-methyl)-methlonine as methyl donor. Thirty-nine individuals were included in the study. A cut-off value of 9 U/ml pRBC was used to distinguish intermediate TPMT enzyme activity from high TPMT enzyme activity. Results: Sequencing of the real-time RT-PCR amplicon proved that the method was specific for the TPMT cDNA, without co-amplification of the highly similar TPMT processed pseudogene. The intra-assay coefficients of variation (CVs), as determined by the threshold cycle, were 0.7% for TPMT and 0.5% for huCYC. The interassay CVs were 1.5% for TPMT and 4.0% for huCYC. The intra- and interassay CVs, as determined by the TPMT/huCYC ratio, were 8.6% and 25%, respectively. There was a statistically significant correlation between TPMT enzyme activity and mRNA level in blood cells from individuals with an enzyme activity above 9 U/ml pRBC (rs = 0.66, P = 0.0001). However, we did not find any statistically significant correlation in individuals with lower enzyme activity or when analysing the whole population. Conclusion: We present a specific and robust real-time RT-PCR method for quantifying TPMT gene expression. The method may be used for studies on TPMT gene regulation.

  • 22.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Berg, Jonathan
    City Hospital, England .
    Duley, John
    University of Queensland, Australia .
    Evans, William E.
    St Jude Childrens Research Hospital, TN USA .
    Kennedy, Martin A:
    University of Otago, New Zealand .
    Lennard, Lynne
    University of Sheffield, England .
    Marinaki, Tony
    Guys and St Thomas Hospital, England .
    McLeod, Howard L.
    University of N Carolina, NC USA .
    Relling, Mary V.
    St Jude Childrens Research Hospital, TN USA .
    Schaeffeler, Elke
    Dr Margarete Fischer Bosch Institute Clin Pharmacol, Germany .
    Schwab, Matthias
    Dr Margarete Fischer Bosch Institute Clin Pharmacol, Germany .
    Weinshilboum, Richard
    Mayo Clin, MN USA .
    Yeoh, Allen E J
    National University of Singapore, Singapore .
    McDonagh, Ellen M.
    Stanford University, CA USA .
    Hebert, Joan M.
    Stanford University, CA USA .
    Klein, Teri E.
    Stanford University, CA USA .
    Coulthard, Sally A.
    Newcastle University, England .
    Nomenclature for alleles of the thiopurine methyltransferase gene2013In: Pharmacogenetics & Genomics, ISSN 1744-6872, E-ISSN 1744-6880, Vol. 23, no 4, p. 242-248Article, review/survey (Refereed)
    Abstract [en]

    The drug-metabolizing enzyme thiopurine methyltransferase (TPMT) has become one of the best examples of pharmacogenomics to be translated into routine clinical practice. TPMT metabolizes the thiopurines 6-mercaptopurine, 6-thioguanine, and azathioprine, drugs that are widely used for treatment of acute leukemias, inflammatory bowel diseases, and other disorders of immune regulation. Since the discovery of genetic polymorphisms in the TPMT gene, many sequence variants that cause a decreased enzyme activity have been identified and characterized. Increasingly, to optimize dose, pretreatment determination of TPMT status before commencing thiopurine therapy is now routine in many countries. Novel TPMT sequence variants are currently numbered sequentially using PubMed as a source of information; however, this has caused some problems as exemplified by two instances in which authors articles appeared on PubMed at the same time, resulting in the same allele numbers given to different polymorphisms. Hence, there is an urgent need to establish an order and consensus to the numbering of known and novel TPMT sequence variants. To address this problem, a TPMT nomenclature committee was formed in 2010, to define the nomenclature and numbering of novel variants for the TPMT gene. A website (http://www.imh.liu.se/tpmtalleles) serves as a platform for this work. Researchers are encouraged to submit novel TPMT alleles to the committee for designation and reservation of unique allele numbers. The committee has decided to renumber two alleles: nucleotide position 106 (Gandgt;A) from TPMT*24 to TPMT*30 and position 611 (Tandgt;C, rs79901429) from TPMT*28 to TPMT*31. Nomenclature for all other known alleles remains unchanged. Pharmacogenetics and Genomics 23: 242-248

  • 23.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Haglund, Sofie
    Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences.
    Almer, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Taipalensuu, Jan
    Division of Research and Development in Laboratory Medicine, Ryhov County Hospital, Jönköping.
    Hertervig, Erik
    dDepartment of Medicine, Lund University Hospital, Lund.
    Lyrenäs, Ebbe
    Department of Medicine, Blekinge County Hospital, Karlskrona.
    Söderkvist, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Identification of two novel sequence variants affecting thiopurine methyltransferase enzyme activity2004In: Pharmacogenetics, ISSN 0960-314X, E-ISSN 1473-561X, Vol. 14, no 4, p. 261-265Article in journal (Refereed)
    Abstract [en]

    The polymorphic enzyme thiopurine methyltransferase (TPMT) is involved in the methylation of thiopurines. On comparing the phenotype with the genotype in Swedish patients with inflammatory bowel disease and healthy individuals, we found two discordant cases with low TPMT enzyme activity (0.3 and 0.4 U/ml packed red blood cells (pRBC). Genotyping by pyrosequencing revealed that they carried the nucleotide substitutions 460G>A and 719A>G, giving two possible genotypes (TPMT*1/*3A or TPMT*3B/ *3C). DNA sequencing of exon III to X was performed in the patients and their parents. We identified an A>G transition in the start codon (exon III, 1A>G, Met>Val, TPMT*14) in one of the patients and her father (6.3 U/ml pRBC). The mother in this family carried the 460G>A and 719A>G nucleotide substitutions (TPMT*3A, 5.0 U/ml pRBC). In the second family, sequencing revealed a G>A transition in the acceptor splice site in intron VII/exon VIII (IVS7 - 1G>A, TPMT*15) in the patient and his mother (6.9 U/ml pRBC). His father was genotyped as TPMT*1/*3A (6.0 U/ml pRBC). Hence, we report the identification of two novel sequence variants, present in highly conserved nucleotide positions of the human TPMT gene, resulting in a loss of enzyme activity.

  • 24.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Hindorf, U
    Almer, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Söderkvist, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Ström, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Hjortswang, Henrik
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    No induction of thiopurine methyltransferase during thiopurine treatment in inflammatory bowel disease2006In: Nucleosides, Nucleotides & Nucleic Acids, ISSN 1525-7770, E-ISSN 1532-2335, Vol. 25, no 9-11, p. 1033-1037Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to follow, during standardized initiation of thiopurine treatment, thiopurine methyltransferase (TPMT) gene expression and enzyme activity and thiopurine metabolite concentrations, and to study the role of TPMT and ITPA 94C > A polymorphisms for the development of adverse drug reactions. Sixty patients with ulcerative colitis or Crohn's disease were included in this open and prospective multi-center study. Thiopurine naïve patients were prescribed azathioprine (AZA), patients previously intolerant to AZA received 6-mercaptopurine (6-MP). The patients followed a predetermined dose escalation schedule, reaching target dose at Week 3, 2.5 and 1.25 mg/kg body weight for AZA and 6-MP, respectively. The patients were followed every week during Weeks 1-8 from baseline and then every 4 weeks until 20 weeks. TPMT activity and thiopurine metabolites were determined in erythrocytes, TPMT and ITPA genotypes, and TPMT gene expression were determined in whole blood. One homozygous TPMT-deficient patient was excluded. Five non compliant patients were withdrawn during the first weeks. Twenty-seven patients completed the study per protocol, 27 patients were withdrawn because of adverse events. Sixty-seven percent of the withdrawn patients tolerated thiopurines at a lower dose at Week 20. There was no difference in baseline TPMT enzyme activity between individuals completing the study and those withdrawn for adverse events (p = 0.45). A significant decrease in TPMT gene expression (TPMT/huCYC ratio, p = 0.02) was found, however TPMT enzyme activity did not change. TPMT heterozygous individuals had a lower probability of remaining in the study on the predetermined dose (p = 0.039). The ITPA 94C > A polymorphism was not predictive of adverse events (p = 0.35). Copyright © Taylor & Francis Group, LLC.

  • 25.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Hindorf, Ulf
    Lund.
    Almer, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Thiopurines in inflammatory bowel disease - The role of pharmacogenetics and therapeutic drug monitoring2006In: Current Pharmacogenomics, ISSN 1570-1603, Vol. 4, no 4, p. 285-300Article in journal (Refereed)
    Abstract [en]

    Pharmacogenetics represents the study of variability in drug response due to genetic variations. Inflammatory bowel disease (IBD, i.e. primarily Crohn's disease and ulcerative colitis) is characterized by a chronic or relapsing inflammation of the digestive tract. The thiopurines 6-mercaptopurine (6-MP) and azathioprine (AZA), an imidazol derivative and pro-drug of 6-MP, are widely used in IBD, particularly in Crohn's disease. The metabolism of thiopurines is complex and individually variable. Thiopurine methyltransferase (TPMT) is a key enzyme in this metabolism and exhibits a genetic variability due to a number of variant alleles coding for a defective enzyme. The formation of biologically active thioguanine nucleotides (TGN) and methylated metabolites may vary considerably due to the TPMT activity. Patients with decreased TPMT activity are at increased risk of developing severe side effects if treated with conventional thiopurine doses, due to the accumulation of toxic metabolites. Determination of the TPMT phenotype or genotype is often used to identify individuals with increased risk for adverse events. Twenty-one variant TPMT alleles have been described, of which three are more common than the others. An association between inosine triphosphate pyrophosphatase polymorphisms and adverse events during thiopurine treatment has also been proposed. In this review, the clinical value of TPMT status determination and pharmacological monitoring of thiopurine metabolites are discussed as well as the increased interest in the use of 6-thioguanine, a thiopurine with a less complex metabolism, as an alternative for patients who do not tolerate AZA or 6-MP. It can be concluded that TPMT determination before start of thiopurine therapy is of value to identify individuals with increased risk for adverse reactions due to genetic enzyme deficiency. However, large prospective studies are still needed to evaluate the true benefit of monitoring thiopurine metabolites during thiopurine treatment. © 2006 Bentham Science Publishers Ltd.

  • 26.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Hindorf, Ulf
    Lund.
    Almer, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Söderkvist, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Ström, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Hjortswang, Henrik
    Linköping University, Department of Molecular and Clinical Medicine.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    No induction of thiopurine methyltransferase (TPMT) during thiopurine treatment in IBD2005In: 10th Symposium European Society for the Study of Purine and Pyrimidine Metabolism in Man,2005, 2005Conference paper (Refereed)
  • 27.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Hindorf, Ulf
    Lund.
    Almer, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Söderkvist, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Ström, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Hjortswang, Henrik
    Linköping University, Department of Molecular and Clinical Medicine.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    No induction of thiopurine methyltransferase (TPMT) during thiopurine treatment in IBD2005In: 13th United European Gastroenterology week,2005, Stuttgart: Georg Thieme Verlag KG , 2005, p. A173-Conference paper (Refereed)
  • 28.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Almer, Sven
    Karolinska institutet - Institutionen för medicin Linköping, Sweden Karolinska institutet - Institutionen för medicin Linköping, Sweden..
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Östergötlands Läns Landsting, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Nyttan av farmakogenetik för en mer individualiserad behandling: Exemplet tiopuriner vid inflammatorisk tarmsjukdom och barnleukemi2015In: Läkartidningen, ISSN 0023-7205, E-ISSN 1652-7518, Vol. 112, p. 1229-1233, article id DF7LArticle in journal (Refereed)
    Abstract [en]

    Thiopurines are chemotherapeutic drugs used for treatment of inflammatory bowel diseases and childhood leukemia. Thiopurine methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism of thiopurines. Individuals lacking TPMT are at increased risk for severe side effects when treated with conventional doses of thiopurines. A research group at the division of drug research at Linköping University is studying thiopurine pharmacogenetics. Since the year 2000, the lab has determined the TPMT status in over 12000 individuals, as an aid to decide thiopurine doses before starting treatment. New knowledge of how genetic factors influence thiopurine treatment effect are anticipated to improve the possibilities for individualization of thiopurine therapy.

  • 29.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Pettersson, Birgitta
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Almer, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, MKC - Medicin och kirurgicentrum, GE: gastromed.
    Albertioni, Freidoun
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Söderhäll, Stefan
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    The role of TPMT polymorphism for thiopurine metabolism and clinical effects.2001In: European society for the study of purine and pyrimidine metabolism in Man.,2001, 2001Conference paper (Refereed)
  • 30.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Skoglund, Karin
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Hertervig, Erik
    Gastroenterologi, Lunds universitetssjukhus.
    Söderkvist, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. 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 for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology UHL.
    Explaining TPMT genotype/phenotype discrepancy by identification of a novel sequence variant, TPMT*262008Conference paper (Refereed)
  • 31.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Skoglund, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Karlgren, Anna
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology.
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Söderkvist, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Kidhall, Irene
    Danderyds sjukhus.
    Almer, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Explaining TPMT genotype/phenotype discrepancy by haplotyping of TPMT*3A and identification of a novel sequence variant, TPMT*232007In: Pharmacogenetics and Genomics, ISSN 1744-6872, Vol. 17, no 10, p. 891-895Article in journal (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. © 2007 Lippincott Williams & Wilkins, Inc.

  • 32.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Wagner, Agnieszka
    Blekinge Hospital, Sweden .
    Hindorf, Ulf
    Skåne University Hospital, Sweden .
    A skewed thiopurine metabolism is a common clinical phenomenon that can be successfully managed with a combination of low-dose azathioprine and allopurinol2013In: Journal of Crohn's & Colitis, ISSN 1873-9946, E-ISSN 1876-4479, Vol. 7, no 6, p. 510-513Article in journal (Refereed)
    Abstract [en]

    Background and aims: A skewed thiopurine metabolism is a phenomenon associated with both poor treatment response and toxicity. Our aim was to evaluate the frequency of this phenomenon and the relationship to thiopurine methyltransferase (TPMT) function. less thanbrgreater than less thanbrgreater thanMethods: All thiopurine metabolite measurements in adult patients (n=4033) between January 2006 and April 2012 were assessed to evaluate the occurrence of a skewed metabolism and the relationship to TPMT genotype and activity. less thanbrgreater than less thanbrgreater thanResults: A skewed metabolism was observed in 14% of all patients. It only developed in patients with a normal TPMT genotype, but was observed at all TPMT activity levels within the normal range (9.1-24.2 U/ml RBC). Two cases that illustrate typical clinical scenarios of a skewed metabolism and the effect of combination treatment with low-dose azathioprine and allopurinol are presented. less thanbrgreater than less thanbrgreater thanConclusions: A skewed metabolism is a common clinical phenomenon in patients with a normal TPMT function, which can develop at all TPMT activity levels within the normal range. We suggest that metabolite measurements should be considered in patients not responding to treatment and in those with hepatotoxicity or myelotoxicity in order to detect a skewed metabolism, since this phenomenon can be successfully managed by a combination of low-dose azathioprine and allopurinol.

  • 33.
    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.

  • 34.
    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.

  • 35.
    Niklasson, Markus
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Helander, Sara
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Roth, Marie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Zimdahl Kahlin, Anna
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Robust and convenient analysis of protein thermal and chemical stability2015In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 24, no 12, p. 2055-2062Article in journal (Refereed)
    Abstract [en]

    We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard assumptions and equations applied to two-state and various types of three-state denaturation models in order to determine thermodynamic parameters. It can analyze denaturation monitored by both circular dichroism and fluorescence spectroscopy and is extremely flexible in terms of input format. Furthermore, it is intuitive and easy to use because of the graphical user interface and extensive documentation. As illustrated by the examples herein, CDpal should be a valuable tool for analysis of protein stability.

  • 36.
    Ping Heidi Iu, Yan
    et al.
    Queen Elizabeth Hospital, Peoples R China.
    Helander, Sara
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Zimdahl Kahlin, Anna
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Wah Cheng, Chun
    Queen Elizabeth Hospital, Peoples R China.
    Chung Shek, Chi
    Queen Elizabeth Hospital, Peoples R China.
    Ho Leung, Moon
    Queen Elizabeth Hospital, Peoples R China.
    Wallner, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    One amino acid makes a difference-Characterization of a new TPMT allele and the influence of SAM on TPMT stability2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 46428Article in journal (Refereed)
    Abstract [en]

    Thiopurine induced toxicity is associated with defects in the thiopurine methyltransferase (TPMT) gene. TPMT is a polymorphic enzyme, with most of the single nucleotide polymorphisms (SNPs) causing an amino acid change, altering the enzymatic activity of the TPMT protein. In this study, we characterize a novel patient allele c.719A amp;gt; C, named TPMT*41, together with the more common variant *3C c.719A amp;gt; G, resulting in an amino acid shift at tyrosine 240 to serine, p.Y240S and cysteine, p.Y240C respectively. We show that the patient heterozygote for c.719A amp;gt; C has intermediate enzymatic activity in red blood cells. Furthermore, in vitro studies, using recombinant protein, show that TPMT p.Y240S is less stable than both TPMTwt and TPMT p.Y240C. The addition of SAM increases the stability and, in agreement with Isothermal Titration Calorimetry (ITC) data, higher molar excess of SAM is needed in order to stabilize TPMT p.Y240C and TPMT p.Y240S compared to TPMTwt. Molecular dynamics simulations show that the loss of interactions is most severe for Y240S, which agrees with the thermal stability of the mutations. In conclusion, our study shows that SAM increases the stability of TPMT and that changing only one amino acid can have a dramatic effect on TPMT stability and activity.

  • 37.
    Skoglund, Karin
    et al.
    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.
    Almér, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Endocrinology and Gastroenterology UHL.
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology UHL.
    Söderkvist, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Low expression of TPMT wild type alleles in a patient with absent TPMT activity2008Conference paper (Other academic)
  • 38.
    Skoglund, Karin
    et al.
    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.
    Almér, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Endocrinology and Gastroenterology UHL.
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology UHL.
    Söderkvist, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Low Expression of TPMT wild type alleles in a patient with absent TPMT activity2008Conference paper (Other academic)
  • 39.
    Skoglund, Karin
    et al.
    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.
    Karlgren, Anna
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Söderkvist, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. 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 for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology UHL.
    Kidhall, Irene
    Div of Gastroenterology and Hepatology, Danderyd Hospital.
    Almér, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Endocrinology and Gastroenterology UHL.
    Explaining TPMT Genotype/Phenotype Discrepancy by Identification of a Novel Sequence Variant, TPMT*232007Conference paper (Other academic)
  • 40.
    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.

  • 41.
    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.

  • 42.
    Zimdahl Kahlin, Anna
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Helander, Sara
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Chemistry. Linköping University, Faculty of Medicine and Health Sciences.
    Skoglund, Karin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Söderkvist, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Comprehensive study of thiopurine methyltransferase genotype, phenotype, and genotype-phenotype discrepancies in Sweden2019In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 164, p. 263-272Article in journal (Refereed)
    Abstract [en]

    Thiopurines are widely used in the treatment of leukemia and inflammatory bowel diseases. Thiopurine metabolism varies among individuals because of differences in the polymorphic enzyme thiopurine methyltransferase (TPMT, EC 2.1.1.67), and to avoid severe adverse reactions caused by incorrect dosing it is recommended that the patients TPMT status be determined before the start of thiopurine treatment. This study describes the concordance between genotyping for common TPMT alleles and phenotyping in a Swedish cohort of 12,663 patients sampled before or during thiopurine treatment. The concordance between TPMT genotype and enzyme activity was 94.5%. Compared to the genotype, the first measurement of TPMT enzyme activity was lower than expected for 4.6% of the patients. Sequencing of all coding regions of the TPMT gene in genotype/phenotype discrepant individuals led to the identification of rare and novel TPMT alleles. Fifteen individuals (0.1%) with rare or novel genotypes were identified, and three TPMT alleles (TPMT*42, *43, and *44) are characterized here for the first time. These 15 patients would not have been detected as carrying a deviating TPMT genotype if only genotyping of the most common TPMT variants had been performed. This study highlights the benefit of combining TPMT genotype and phenotype determination in routine testing. More accurate dose recommendations can be made, which might decrease the number of adverse reactions and treatment failures during thiopurine treatment.

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