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Haglund, Sofie
Publications (9 of 9) Show all publications
Haglund, S., Zimdahl Kahlin, A., Vikingsson, S., Almér, S. & Söderman, J. (2014). P658 Effects of allopurinol on thiopurine metabolism and gene expression levels in HepG2 cells. In: : . Paper presented at 9th Congress of ECCO - the European Crohn's and Colitis Organisation (pp. 1:S345). , 8
Open this publication in new window or tab >>P658 Effects of allopurinol on thiopurine metabolism and gene expression levels in HepG2 cells
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2014 (English)Conference paper, Published paper (Other academic)
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-117883 (URN)10.1016/S1873-9946(14)60777-7 (DOI)
Conference
9th Congress of ECCO - the European Crohn's and Colitis Organisation
Available from: 2015-05-12 Created: 2015-05-12 Last updated: 2015-05-26
Haglund, S., Almer, S., Peterson, C. & Söderman, J. (2013). Gene Expression and Thiopurine Metabolite Profiling in Inflammatory Bowel Disease: Novel Clues to Drug Targets and Disease Mechanisms?. PLoS ONE, 8(2)
Open this publication in new window or tab >>Gene Expression and Thiopurine Metabolite Profiling in Inflammatory Bowel Disease: Novel Clues to Drug Targets and Disease Mechanisms?
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 2Article in journal (Refereed) Published
Abstract [en]

Background and Aims

Thiopurines are effective to induce and maintain remission in inflammatory bowel disease (IBD). The methyl thioinosine monophosphate (meTIMP)/6-thioguanine nucleotide (6-TGN) concentration ratio has been associated with drug efficacy. Here we explored the molecular basis of differences in metabolite profiles and in relation to disease activity.

Methods

Transcriptional profiles in blood samples from an exploratory IBD-patient cohort (n = 21) with a normal thiopurine S-methyltransferase phenotype and meTIMP/6-TGN ratios >20, 10.0–14.0 and ≤4, respectively, were assessed by hybridization to microarrays. Results were further evaluated with RT qPCR in an expanded patient cohort (n = 54). Additionally, 30 purine/thiopurine related genes were analysed separately.

Results

Among 17 genes identified by microarray-screening, there were none with a known relationship to pathways of purines/thiopurines. For nine of them a correlation between expression level and the concentration of meTIMP, 6-TGN and/or the meTIMP/6-TGN ratio was confirmed in the expanded cohort. Nine of the purine/thiopurine related genes were identified in the expanded cohort to correlate with meTIMP, 6-TGN and/or the meTIMP/6-TGN ratio. However, only small differences in gene expression levels were noticed over the three different metabolite profiles. The expression levels of four genes identified by microarray screening (PLCB2, HVCN1, CTSS, and DEF8) and one purine/thiopurine related gene (NME6) correlated significantly with the clinical activity of Crohn’s disease. Additionally, 16 of the genes from the expanded patient cohort interacted in networks with candidate IBD susceptibility genes.

Conclusions

Seventeen of the 18 genes which correlated with thiopurine metabolite levels also correlated with disease activity or participated in networks with candidate IBD susceptibility genes involved in processes such as purine metabolism, cytokine signaling, and functioning of invariant natural killer T cells, T cells and B cells. Therefore, we conclude that the identified genes to a large extent are related to drug targets and disease mechanisms of IBD.

Place, publisher, year, edition, pages
Public Library of Science, 2013
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-90198 (URN)10.1371/journal.pone.0056989 (DOI)000315186000065 ()
Note

Funding Agencies|Futurum - the Academy for Healthcare||County Council Jonkoping||Medical Research Council of Southeast Sweden (FORSS)||Swedish Society of Medicine||County Council of Ostergotland||Swedish Cancer Society||Swedish Childhood Cancer Foundation||Rut and Richard Juhlins Foundation||Swedish Research Council||

Available from: 2013-03-21 Created: 2013-03-21 Last updated: 2017-12-06
Haglund, S. (2011). Interindividual differences in thiopurine metabolism: studies with focus on inflammatory bowel disease. (Doctoral dissertation). Linköping: Linköping University Electronic Press
Open this publication in new window or tab >>Interindividual differences in thiopurine metabolism: studies with focus on inflammatory bowel disease
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The thiopurines, 6-mercaptopurine and its prodrug azathioprine, are used in the treatment of inflammatory bowel disease, ulcerative colitis and Crohn´s disease. The main active metabolites are the phosphorylated thioguanine nucleotides (6-TGNs) and methylated thioinosine monophosphate (meTIMP). Both groups contribute to the immunomodulatory effects. About 30-40% of patients fail to benefit from thiopurine treatment. A well-known cause of adverse reactions is decreased or absent thiopurine S-methyltransferase (TPMT) activity. Low TPMT activity is inherited as an autosomal codominant recessive trait and is present in approximately 10% of the population. Although several clinical issues can be solved from determination of TPMT activity, there are cases where it is not possible. In Sweden approximately 25% of IBD-patients display suboptimal 6-TGN concentrations and unexpectedly high concentrations of meTIMP despite a normal TPMT activity. A high meTIMP/6-TGN concentration ratio has been associated with both unresponsiveness to therapy and emergence of adverse reactions. Inosine 5’-monophosphate dehydrogenase (IMPDH) may constitute a candidate gene to explain this metabolite profile, as it is strategically positioned in the metabolic pathway of thiopurines where it competes with TPMT for their common substrate 6-TIMP.

In paper I a pyrosequencing method was developed for genotyping of at that time all known genetic variants of TPMT. The concordance between genotype and phenotype in 30 individuals was 93%. The allele frequencies of TPMT*3A, *3B, *3C and *2 in a Swedish background population (n=800) were in agreement with those in other Caucasian or European populations. In Paper II-IV we explored the molecular basis of different metabolite profiles, i.e. low, normal and high meTIMP/6-TGN concentration ratios. The activity of IMPDH was measured in mononuclear cells (MNC). Patients with high metabolite ratios had lower IMPDH activity than patients with normal or low ratios, explained by an inverse correlation to red blood cells concentration of meTIMP. No correlation to 6-TGN was observed. Downregulation of IMPDH activity in HEK293 cells with genetically engineered TPMT activity was associated with an increase in meTIMP, but unexpectedly also of 6-TGN, irrespective of the TPMT status. These results suggest effects of pharmacogenes other than TPMT and IMPDH. A whole genome expression analysis was performed, (1) to identify new candidate genes that could explain differences in metabolite profiles, and (2) to study genes with known associations to the metabolic pathway of (thio)purines. The whole genome expression analysis did not identify any significant group differences. In analysis of the thiopurine related genes, three clusters of co-regulated genes were defined. A co-operation between expression levels of SLC29A1 and NT5E in explaining the meTIMP/6-TGN concentration ratio was observed, and individually SLC29A1 and NT5E correlated to 6-TGN and meTIMP, respectively.

Pysosequencing is a convenient and flexible method which is now run in parallel to phenotyping in our laboratory. Our results also illustrate the complexity of the thiopurine metabolism and suggest that differences between metabolite profiles are explained either by interactions between several genes, each with a small contribution, or at the post-transcriptional level. Search for more precise tools to explain differences in metabolite profiles is needed. Furthermore, in order to investigate small effects it is necessary to analyse metabolite concentrations and gene expression levels, as well as enzyme activities in the target cells of therapy (MNC).

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2011. p. 96
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1231
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-66434 (URN)978-91-7393-213-4 (ISBN)
Public defence
2011-04-15, Aulan, Länssjukhuset Ryhov, Jönköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2011-03-15 Created: 2011-03-15 Last updated: 2012-03-22Bibliographically approved
Haglund, S., Vikingsson, S., Söderman, J., Hindorf, U., Grännö, C., Danelius, M., . . . Almer, S. (2011). The Role of Inosine-5'-Monophosphate Dehydrogenase in Thiopurine Metabolism in Patients With Inflammatory Bowel Disease. Therapeutic Drug Monitoring, 33(2), 200-208
Open this publication in new window or tab >>The Role of Inosine-5'-Monophosphate Dehydrogenase in Thiopurine Metabolism in Patients With Inflammatory Bowel Disease
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2011 (English)In: Therapeutic Drug Monitoring, ISSN 0163-4356, E-ISSN 1536-3694, Vol. 33, no 2, p. 200-208Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:: There is a large interindividual variability in thiopurine metabolism. High concentrations of methylthioinosine-5'-monophosphate (meTIMP) and low concentrations of 6-thioguanine nucleotides (6-TGNs) have been associated with a lower response rate and an increased risk of adverse events. In this study, the role of inosine-5'-monophosphate dehydrogenase (IMPDH) for differences in metabolite patterns of thiopurines was investigated.

METHODS:: IMPDH activity and thiopurine metabolite concentrations were determined in patients with inflammatory bowel disease and a normal thiopurine methyltransferase (TPMT) phenotype and meTIMP/6-TGN concentration ratio > 20 (n = 26), in patients with a metabolite ratio ≤20 (n = 21), in a subgroup with a metabolite ratio <4 (n = 6), and in 10 patients with reduced TPMT activity. In vitro studies were conducted on human embryonic kidney cells (HEK293) with genetically engineered IMPDH and TPMT activities.

RESULTS:: Patients with metabolite ratios >20 had lower IMPDH activity than those with ratios ≤20 (P < 0.001). Metabolic ratios >20 were only observed in patients with normal TPMT activity. Downregulation of IMPDH activity in HEK293 cells was associated with an increase in the concentration of meTIMP (fold change: 17 up to 93, P < 0.001) but, unexpectedly, also of 6-thioguanosine monophosphate (fold change: 2.6 up to 5.0, P < 0.001).

CONCLUSIONS:: These data question the general view of IMPDH as the rate-limiting enzyme in the phosphorylation of thiopurines. Investigations of other mechanisms are needed to more fully explain the various metabolite patterns and outcomes in patients under treatment.

Place, publisher, year, edition, pages
Lippincott Williams & Wilkins, 2011
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-66431 (URN)10.1097/FTD.0b013e31820b42bb (DOI)000288498100010 ()21311411 (PubMedID)
Available from: 2011-03-15 Created: 2011-03-15 Last updated: 2017-12-11Bibliographically approved
Rajani, R., Haglund, S., Bergquist, A., Melin, T., Friis-Liby, I., Verbaan, H., . . . Almer, S. (2010). High prevalence of the germline JAK2 46/1 haplotype and V617-mutationin Swedish patients with Budd-Chiari syndrome and Portal Vein Thrombosis. , 52(4)
Open this publication in new window or tab >>High prevalence of the germline JAK2 46/1 haplotype and V617-mutationin Swedish patients with Budd-Chiari syndrome and Portal Vein Thrombosis
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2010 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Background & Aims: To determine the prevalence of the somatic JAK2 V617F mutation and distribution of the germline JAK2 46/1 haplotype in Budd-Chiari Syndrome (BCS) and portal vein thrombosis (PVT).

Methods: Real-time PCR was performed to genotype for the JAK2V 617F mutation and the 46/1 haplotype (tag-SNPs rs12343867, T>C and rs12340895, C>G) in blood samples of 19 BCS and 91 PVT patients (without intra-abdominal malignancy), and 283 controls from a background population.

Results: The prevalence of JAK2 V617F-mutation was 63% in BCS and 14% in PVT patients. 10% in BCS and 2% in PVT had V617F negative MPD. Conversely, V617F positive subjects without known MPD was found in 5% of the BCS and in 1% of PVT patients. The frequency of the JAK2 46/1 haplotype was significantly higher in BCS (53%) and PVT (36%) patients compared to controls (27%) (p=0.02; OR=3.0; 95% CI 1.5-5.9 and OR=1.51; 95% CI 1.1-2.1, respectively). In PVT patients the JAK2 haplotype was highly enriched in non-cirrhotic patients (41%) (p <0.01 ; OR=1.8; 95% CI 1.2-2.6) but not in cirrhotic patients (23%) (p=0.53 ; OR= 0.8; 95% CI 0.4-1.7). An increased JAK2 46/1 haplotype frequency was evident only in V617F mutation positive patients.

Conclusions: The prevalence of JAK2 V617F was high in BCS (63%) and non-cirrhotic PVT (14%), facilitating detection of latent MPD. A negative result dose not rule out MPD. The occurrence of the JAK2 46/1 haplotype was significantly higher in V617F mutation positive patients but not in mutation negative patients, suggesting that the haplotype may not have an independent role separated from the V617F mutation in BCS and PVT patients.

Keywords
Budd-Chiari syndrome; Portal vein thrombosis; Myeloproliferative disorders; JAK2 V617F mutation; JAK2 46/1 haplotype; Thrombophilia; Liver cirrhosis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-68726 (URN)
Available from: 2011-05-31 Created: 2011-05-31 Last updated: 2011-05-31Bibliographically approved
Haglund, S., Taipalensuu, J., Peterson, C. & Almer, S. (2008). IMPDH activity in thiopurine-treated patients with inflammatory bowel disease - Relation to TPMT activity and metabolite concentrations. British Journal of Clinical Pharmacology, 65(1), 69-77
Open this publication in new window or tab >>IMPDH activity in thiopurine-treated patients with inflammatory bowel disease - Relation to TPMT activity and metabolite concentrations
2008 (English)In: British Journal of Clinical Pharmacology, ISSN 0306-5251, E-ISSN 1365-2125, Vol. 65, no 1, p. 69-77Article in journal (Refereed) Published
Abstract [en]

AIMS: Azathioprine and 6-mercaptopurine are steroid-sparing drugs used in inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of importance for thiopurine metabolism and occurrence of adverse events. The role of other thiopurine-metabolizing enzymes is less well known. This study investigated the role of inosine-5′- monophosphate dehydrogenase (IMPDH), which is a key enzyme in the de novo synthesis of guanine nucleotides and also strategically positioned in the metabolic pathway of thiopurines. METHODS: IMPDH was measured in 100 healthy blood donors. IMPDH, TPMT and metabolite concentrations were studied in 50 patients with IBD on stable thiopurine therapy. IMPDH activity was measured in peripheral blood mononuclear cells. TPMT activity, 6-methylthioinosine 5′-monophosphate (meTIMP) and 6-thioguanine nucleotide (6-TGN) concentrations were measured in red blod cells, which is the current practice in clinical monitoring of thiopurines. Enzyme activities were related to metabolite concentrations and clinical characteristics. RESULTS: A wide range of IMPDH activity was observed both in healthy blood donors (median 13.1, range 4.7-24.2 nmol mg-1 protein h-1) and IBD patients (median 14.0, range 7.0-21.7). There was a negative correlation between IMPDH activity and dose-normalized meTIMP concentrations (rs = -0.31, P = 0.03), but no evident correlation to 6-TGN concentration or the meTIMP/6-TGN ratio. There were no significant correlations between TPMT activity and metabolite concentrations. CONCLUSION: Even though the meTIMP concentrations correlated inversely to the IMPDH activity, the role of IMPDH in balancing the formation of methylated and phosphorylated metabolites was not evident. Taken together, the results give cause to question established opinions about thiopurine metabolism. © 2007 The Authors.

Keywords
6-Mercaptopurine/*pharmacology Adult Aged Aged, 80 and over Azathioprine/*pharmacology Biological Markers Female Humans IMP Dehydrogenase/genetics/*metabolism Immunosuppressive Agents/*pharmacology Inflammatory Bowel Diseases/*drug therapy Male Methyltran
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-43268 (URN)10.1111/j.1365-2125.2007.02985.x (DOI)73262 (Local ID)73262 (Archive number)73262 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13Bibliographically approved
Haglund, S., Almer, S., Peterson, C. & Söderman, J. (2008). Pharmacotranscriptomics in thiopurine treated IBD patients with different metabolite profiles. , 65(1)
Open this publication in new window or tab >>Pharmacotranscriptomics in thiopurine treated IBD patients with different metabolite profiles
2008 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Thiopurine drugs are used to induce and maintain remission in inflammatory bowel disease. The methyl thioinosine monophosphate (meTIMP)/6-thioguanine nucleotide (6-TGN) concentration ratio has been related to drug response and adverse reactions. Here we investigated for differences in gene expression levels between patients with different metabolite profiles.

Methods: Transcriptional profiles in blood samples from an exploratory patient cohort (n=21) comprising three groups; patients with normal thiopurine S-methyltransferase phenotype and meTIMP/6-TGN concentration ratio >20, ratio 10.0-14.0 and ratio ≤4, respectively, were assessed by hybridization to microarrays. Results were further evaluated with reverse transcription qPCR [exploratory and a validation cohort of patients (n=33)]. Additionally, known genes of the thiopurine metabolic pathway were analysed separately.

Results: The whole genome expression analysis did not identify any significant differences between metabolite profiles. Analysis of thiopurine related genes revealed a large interindividual variation in gene expression, but only small differences between metabolite profiles. Three clusters of co-regulated genes were defined based on correlations between gene expression levels. The concentration of meTIMP correlated to the expression of NT5E (rs = 0.33, P = 0.02) and TPMT (rs = - 0.37, P = 0.007). The concentration of 6-TGN correlated to the expression of HPRT1 (rs = - 0.31, P = 0.03) and SLC29A1 (rs = 0.33, P = 0.02). With the exception of SLC29A1, these genes belonged to the same cluster of genes.

Conclusions: Our results illustrates the complexity of the thiopurine metabolism and suggest that differences between metabolite profiles are explained either by interactions between several genes, each with a small contribution, or at the post-transcriptional level. Search for more precise tools in order to explain differences in metabolite profiles is needed.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-66432 (URN)
Available from: 2011-03-15 Created: 2011-03-15 Last updated: 2011-03-15Bibliographically approved
Lindqvist Appell, M., Haglund, S., Almer, S., Peterson, C., Taipalensuu, J., Hertervig, E., . . . Söderkvist, P. (2004). Identification of two novel sequence variants affecting thiopurine methyltransferase enzyme activity. Pharmacogenetics, 14(4), 261-265
Open this publication in new window or tab >>Identification of two novel sequence variants affecting thiopurine methyltransferase enzyme activity
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2004 (English)In: Pharmacogenetics, ISSN 0960-314X, E-ISSN 1473-561X, Vol. 14, no 4, p. 261-265Article in journal (Refereed) Published
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.

Keywords
drug metabolizing enzyme, polymorphism, thiopurine methyltransferase
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-23408 (URN)10.1097/00008571-200404000-00006 (DOI)2855 (Local ID)2855 (Archive number)2855 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13
Haglund, S., Lindqvist Appell, M., Almer, S., Peterson, C. & Taipalensuu, J. (2004). Pyrosequencing of TPMT Alleles in a General Swedish Population and in Patients with Inflammatory Bowel Disease. Clinical Chemistry, 50(2), 288-295
Open this publication in new window or tab >>Pyrosequencing of TPMT Alleles in a General Swedish Population and in Patients with Inflammatory Bowel Disease
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2004 (English)In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 50, no 2, p. 288-295Article in journal (Refereed) Published
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-45829 (URN)10.1373/clinchem.2003.023846 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
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