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Lin J, Oludare A, Jung H. Connecting dots between nucleotide biosynthesis and DNA lesion repair/bypass in cancer. Biosci Rep 2024; 44:BSR20231382. [PMID: 39189649 PMCID: PMC11427732 DOI: 10.1042/bsr20231382] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2024] [Revised: 08/01/2024] [Accepted: 08/15/2024] [Indexed: 08/28/2024] Open
Abstract
Purine and pyrimidine nucleotides are crucial building blocks for the survival of cells, and there are layers of pathways to make sure a stable supply of them including de novo nucleotide biosynthesis. Fast-growing cells including cancer cells have high demand for nucleotide, and they highly utilize the nucleotide biosynthesis pathways. Due to the nature of the fast-growing cells, they tend to make more errors in replication compared with the normal cells. Naturally, DNA repair and DNA lesion bypass are heavily employed in cancer cells to ensure fidelity and completion of the replication without stalling. There have been a lot of drugs targeting cancer that mimic the chemical structures of the nucleobase, nucleoside, and nucleotides, and the resistance toward those drugs is a serious problem. Herein, we have reviewed some of the representative nucleotide analog anticancer agents such as 5-fluorouracil, specifically their mechanism of action and resistance is discussed. Also, we have chosen several enzymes in nucleotide biosynthesis, DNA repair, and DNA lesion bypass, and we have discussed the known and potential roles of these enzymes in maintaining genomic fidelity and cancer chemotherapy.
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Affiliation(s)
- Jackson C. Lin
- The Division of Medicinal Chemistry, School of Pharmacy, The University of Connecticut, Storrs, Connecticut 06269, U.S.A
| | - Ayobami Oludare
- The Division of Medicinal Chemistry, School of Pharmacy, The University of Connecticut, Storrs, Connecticut 06269, U.S.A
| | - Hunmin Jung
- The Division of Medicinal Chemistry, School of Pharmacy, The University of Connecticut, Storrs, Connecticut 06269, U.S.A
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Averill JR, Lin JC, Jung J, Jung H. Novel insights into the role of translesion synthesis polymerase in DNA incorporation and bypass of 5-fluorouracil in colorectal cancer. Nucleic Acids Res 2024; 52:4295-4312. [PMID: 38416579 PMCID: PMC11077093 DOI: 10.1093/nar/gkae102] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2023] [Revised: 01/12/2024] [Accepted: 02/01/2024] [Indexed: 03/01/2024] Open
Abstract
5-Fluorouracil (5-FU) is the first-line chemotherapeutic agent in colorectal cancer, and resistance to 5-FU easily emerges. One of the mechanisms of drug action and resistance of 5-FU is through DNA incorporation. Our quantitative reverse-transcription PCR data showed that one of the translesion synthesis (TLS) DNA polymerases, DNA polymerase η (polη), was upregulated within 72 h upon 5-FU administration at 1 and 10 μM, indicating that polη is one of the first responding polymerases, and the only TLS polymerase, upon the 5-FU treatment to incorporate 5-FU into DNA. Our kinetic studies revealed that 5-fluoro-2'-deoxyuridine triphosphate (5FdUTP) was incorporated across dA 41 and 28 times more efficiently than across dG and across inosine, respectively, by polη indicating that the mutagenicity of 5-FU incorporation is higher in the presence of inosine and that DNA lesions could lead to more mutagenic incorporation of 5-FU. Our polη crystal structures complexed with DNA and 5FdUTP revealed that dA:5FdUTP base pair is like dA:dTTP in the active site of polη, while 5FdUTP adopted 4-enol tautomer in the base pairs with dG and HX increasing the insertion efficiency compared to dG:dTTP for the incorrect insertions. These studies confirm that polη engages in the DNA incorporation and bypass of 5-FU.
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Affiliation(s)
- Jameson R Averill
- Division of Medicinal Chemistry, School of Pharmacy, University of Connecticut, Storrs, CT 06269, USA
| | - Jackson C Lin
- Division of Medicinal Chemistry, School of Pharmacy, University of Connecticut, Storrs, CT 06269, USA
| | - John Jung
- Division of Medicinal Chemistry, School of Pharmacy, University of Connecticut, Storrs, CT 06269, USA
| | - Hunmin Jung
- Division of Medicinal Chemistry, School of Pharmacy, University of Connecticut, Storrs, CT 06269, USA
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Straube H, Straube J, Rinne J, Fischer L, Niehaus M, Witte CP, Herde M. An inosine triphosphate pyrophosphatase safeguards plant nucleic acids from aberrant purine nucleotides. THE NEW PHYTOLOGIST 2023; 237:1759-1775. [PMID: 36464781 DOI: 10.1111/nph.18656] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/20/2022] [Accepted: 11/22/2022] [Indexed: 06/17/2023]
Abstract
In plants, inosine is enzymatically introduced in some tRNAs, but not in other RNAs or DNA. Nonetheless, our data show that RNA and DNA from Arabidopsis thaliana contain (deoxy)inosine, probably derived from nonenzymatic adenosine deamination in nucleic acids and usage of (deoxy)inosine triphosphate (dITP and ITP) during nucleic acid synthesis. We combined biochemical approaches, LC-MS, as well as RNA-Seq to characterize a plant INOSINE TRIPHOSPHATE PYROPHOSPHATASE (ITPA) from A. thaliana, which is conserved in many organisms, and investigated the sources of deaminated purine nucleotides in plants. Inosine triphosphate pyrophosphatase dephosphorylates deaminated nucleoside di- and triphosphates to the respective monophosphates. ITPA loss-of-function causes inosine di- and triphosphate accumulation in vivo and an elevated inosine and deoxyinosine content in RNA and DNA, respectively, as well as salicylic acid (SA) accumulation, early senescence, and upregulation of transcripts associated with immunity and senescence. Cadmium-induced oxidative stress and biochemical inhibition of the INOSINE MONOPHOSPHATE DEHYDROGENASE leads to more IDP and ITP in the wild-type (WT), and this effect is enhanced in itpa mutants, suggesting that ITP originates from ATP deamination and IMP phosphorylation. Inosine triphosphate pyrophosphatase is part of a molecular protection system in plants, preventing the accumulation of (d)ITP and its usage for nucleic acid synthesis.
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Affiliation(s)
- Henryk Straube
- Department of Molecular Nutrition and Biochemistry of Plants, Leibniz Universität Hannover, Hannover, 30419, Germany
| | - Jannis Straube
- Department of Molecular Plant Breeding, Leibniz Universität Hannover, Hannover, 30419, Germany
| | - Jannis Rinne
- Department of Molecular Nutrition and Biochemistry of Plants, Leibniz Universität Hannover, Hannover, 30419, Germany
| | - Lisa Fischer
- Department of Molecular Nutrition and Biochemistry of Plants, Leibniz Universität Hannover, Hannover, 30419, Germany
| | - Markus Niehaus
- Department of Molecular Nutrition and Biochemistry of Plants, Leibniz Universität Hannover, Hannover, 30419, Germany
| | - Claus-Peter Witte
- Department of Molecular Nutrition and Biochemistry of Plants, Leibniz Universität Hannover, Hannover, 30419, Germany
| | - Marco Herde
- Department of Molecular Nutrition and Biochemistry of Plants, Leibniz Universität Hannover, Hannover, 30419, Germany
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Zamzami MA. Inosine Triphosphate Pyrophosphatase (ITPase): Functions, Mutations, Polymorphisms and Its Impact on Cancer Therapies. Cells 2022; 11:384. [PMID: 35159194 PMCID: PMC8833965 DOI: 10.3390/cells11030384] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2021] [Revised: 01/18/2022] [Accepted: 01/19/2022] [Indexed: 11/16/2022] Open
Abstract
Inosine triphosphate pyrophosphatase (ITPase) is an enzyme encoded by the ITPA gene and functions to prevent the incorporation of noncanonical purine nucleotides into DNA and RNA. Specifically, the ITPase catalyzed the hydrolysis of (deoxy) nucleoside triphosphates ((d) NTPs) into the corresponding nucleoside monophosphate with the concomitant release of pyrophosphate. Recently, thiopurine drug metabolites such as azathioprine have been included in the lists of ITPase substrates. Interestingly, inosine or xanthosine triphosphate (ITP/XTP) and their deoxy analogs, deoxy inosine or xanthosine triphosphate (dITP/dXTP), are products of important biological reactions such as deamination that take place within the cellular compartments. However, the incorporation of ITP/XTP, dITP/dXTP, or the genetic deficiency or polymorphism of the ITPA gene have been implicated in many human diseases, including infantile epileptic encephalopathy, early onset of tuberculosis, and the responsiveness of patients to cancer therapy. This review provides an up-to-date report on the ITPase enzyme, including information regarding its discovery, analysis, and cellular localization, its implication in human diseases including cancer, and its therapeutic potential, amongst others.
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Affiliation(s)
- Mazin A. Zamzami
- Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia;
- Centre of Artificial Intelligence in Precision Medicines, King Abdulaziz University, Jeddah 21589, Saudi Arabia
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Ji D, Stepchenkova EI, Cui J, Menezes MR, Pavlov YI, Kool ET. Measuring deaminated nucleotide surveillance enzyme ITPA activity with an ATP-releasing nucleotide chimera. Nucleic Acids Res 2017; 45:11515-11524. [PMID: 29036687 PMCID: PMC5714213 DOI: 10.1093/nar/gkx774] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2017] [Accepted: 08/23/2017] [Indexed: 01/24/2023] Open
Abstract
Nucleotide quality surveillance enzymes play important roles in human health, by detecting damaged molecules in the nucleotide pool and deactivating them before they are incorporated into chromosomal DNA or adversely affect metabolism. In particular, deamination of adenine moiety in (deoxy)nucleoside triphosphates, resulting in formation of (d)ITP, can be deleterious, leading to DNA damage, mutagenesis and other harmful cellular effects. The 21.5 kDa human enzyme that mitigates this damage by conversion of (d)ITP to monophosphate, ITPA, has been proposed as a possible therapeutic and diagnostic target for multiple diseases. Measuring the activity of this enzyme is useful both in basic research and in clinical applications involving this pathway, but current methods are nonselective and are not applicable to measurement of the enzyme from cells or tissues. Here, we describe the design and synthesis of an ITPA-specific chimeric dinucleotide (DIAL) that replaces the pyrophosphate leaving group of the native substrate with adenosine triphosphate, enabling sensitive detection via luciferase luminescence signaling. The probe is shown to function sensitively and selectively to quantify enzyme activity in vitro, and can be used to measure the activity of ITPA in bacterial, yeast and human cell lysates.
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Affiliation(s)
- Debin Ji
- Department of Chemistry, Stanford University, Stanford, CA 94305, USA
| | - Elena I Stepchenkova
- The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.,Department of Genetics and Biotechnology, Saint-Petersburg State University, St Petersburg, 199034, Russia.,Saint-Petersburg Branch of Vavilov Institute of General Genetics, RAS, St Petersburg, 199034, Russia
| | - Jian Cui
- The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Miriam R Menezes
- Department of Neurosurgery, University of Texas Health Science Center, Houston, TX 77030, USA
| | - Youri I Pavlov
- The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.,Departments of Biochemistry and Molecular Biology; Microbiology and Pathology; Genetics Cell Biology and Anatomy; University of Nebraska Medical Center, Omaha, NE 61818, USA
| | - Eric T Kool
- Department of Chemistry, Stanford University, Stanford, CA 94305, USA
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Rufini S, Ciccacci C, Novelli G, Borgiani P. Pharmacogenetics of inflammatory bowel disease: a focus on Crohn's disease. Pharmacogenomics 2017; 18:1095-1114. [PMID: 28686143 DOI: 10.2217/pgs-2017-0068] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Crohn's disease is an inflammatory bowel disease showing a high heterogeneity in phenotype and a strong genetic component. The treatment is complex, due to different severity of clinical parameters and to the fact that therapies only permit to control symptoms and to induce remission for short periods. Moreover, all categories of drugs present a great interindividual variability both in terms of efficacy and side effects appearance. For this reason, the identification of specific genomic biomarkers involved in drugs response will be of great clinical utility in order to foresee drug's efficacy and to prevent adverse reactions, permitting a more personalized therapeutic approach. In this review, we focus the attention on the pharmacogenetic studies regarding drugs commonly utilized in Crohn's disease treatment.
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Affiliation(s)
- Sara Rufini
- Department of Biomedicine & Prevention, Genetics Unit, University of Rome "Tor Vergata", Rome, Italy
| | - Cinzia Ciccacci
- Department of Biomedicine & Prevention, Genetics Unit, University of Rome "Tor Vergata", Rome, Italy
| | - Giuseppe Novelli
- Department of Biomedicine & Prevention, Genetics Unit, University of Rome "Tor Vergata", Rome, Italy
| | - Paola Borgiani
- Department of Biomedicine & Prevention, Genetics Unit, University of Rome "Tor Vergata", Rome, Italy
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7
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Hwang JJ, Lo CC, Lin CH, Cheng HS, Hung IW, Tsai WJ, Hung CH. Association between IPTA gene polymorphisms and hematological abnormalities in hepatitis C virus-infected patients receiving combination therapy. Gut Liver 2015; 9:214-23. [PMID: 25287171 PMCID: PMC4351029 DOI: 10.5009/gnl14095] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Background/Aims Hematological abnormalities during hepatitis C virus (HCV) combination therapy with pegylated interferon α and ribavirin often necessitate dose reduction. Variants of the ITPA gene have been reported to protect against anemia during the early stages of HCV combination treatments but have also been associated with larger decreases in platelet counts. We aimed to identify the association between specific ITPA gene polymorphisms and hematological abnormalities in patients undergoing HCV combination therapy. Methods In this retrospective study, 175 patients treated with HCV combination therapy were enrolled at St. Martin De Porres Hospital in Taiwan between 2006 and 2012. Two single nucleotide polymorphisms (SNP) within or adjacent to the ITPA gene (rs1127354, rs6051702) were genotyped. We investigated the effect of ITPA gene variants on hematological abnormalities during the therapy. Results The ITPA rs1127354 minor variants were significantly associated with protection against anemia at week 4 (p=1.86×10−6) and with more severe decreases in platelet counts during HCV combination therapy. SNP rs6051702 was not associated with the hemoglobin decline to >3 g/dL at week 4 in our study (p=0.055). Conclusions The ITPA SNP rs1127354 is a useful predictor of ribavirin-induced anemia in Taiwanese patients and may be related to more severe decreases in platelet counts during the early stage of HCV combination therapy.
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Affiliation(s)
- Jow Jyh Hwang
- Section of Gastroenterology and Hepatology, Department of Internal Medicine, St. Martin De Porres Hospital and Graduate Institute of Clinical Medical Sciences, Chang Gung University College of Medicine, Chiayi, Taiwan
| | - Ching Chu Lo
- Section of Gastroenterology and Hepatology, Department of Internal Medicine, St. Martin De Porres Hospital, Chiayi, Taiwan
| | - Chien Hung Lin
- Section of Gastroenterology and Hepatology, Department of Internal Medicine, St. Martin De Porres Hospital, Chiayi, Taiwan
| | - Hsu Sheng Cheng
- Section of Gastroenterology and Hepatology, Department of Internal Medicine, St. Martin De Porres Hospital, Chiayi, Taiwan
| | - I Wen Hung
- Outpatient Nursing Section, St. Martin De Porres Hospital, Chiayi, Taiwan
| | - Wan Ju Tsai
- Graduate Institute of Clinical Medical Sciences, Chang Gung University College of Medicine, Chiayi, Taiwan
| | - Chien Hui Hung
- Graduate Institute of Clinical Medical Sciences, Chang Gung University College of Medicine, Chiayi, Taiwan
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Delvaux N, da Costa VD, da Costa MM, Villar LM, Coelho HSM, Esberard EBC, Flores PP, Brandão-Mello CE, Villela-Nogueira CA, de Almeida AJ, Lampe E. Inosine triphosphatase allele frequency and association with ribavirin-induced anaemia in Brazilian patients receiving antiviral therapy for chronic hepatitis C. Mem Inst Oswaldo Cruz 2015; 110:636-643. [PMID: 26154744 PMCID: PMC4569827 DOI: 10.1590/0074-02760150104] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2015] [Accepted: 06/08/2015] [Indexed: 12/19/2022] Open
Abstract
Inosine triphosphatase (ITPA) single nucleotide polymorphisms (SNPs) are strongly associated with protection against ribavirin (RBV)-induced anaemia in European, American and Asian patients; however, there is a paucity of data for Brazilian patients. The aim of this study was to evaluate the ITPA SNP (rs7270101/rs1127354) frequency in healthy and hepatitis C virus (HCV)-infected patients from Brazil and the association with the development of severe anaemia during antiviral therapy. ITPA SNPs were determined in 200 HCV infected patients and 100 healthy individuals by sequencing. Biochemical parameters and haemoglobin (Hb) levels were analysed in 97 patients who underwent antiviral therapy. A combination of AArs7270101+CCrs1127354 (100% ITPase activity) was observed in 236/300 individuals. Anaemia was observed in 87.5% and 86.2% of treated patients with AA (rs7270101) and CC genotypes (rs1127354), respectively. Men with AA (rs7270101) showed a considerable reduction in Hb at week 12 compared to those with AC/CC (p = 0.1475). In women, there was no influence of genotype (p = 0.5295). For rs1127354, men with the CC genotype also showed a sudden reduction in Hb compared to those with AC. Allelic distribution of rs7270101 and rs1127354 shows high rates of the genotypes AA and CC, respectively, suggesting that the study population had a great propensity for developing RBV-induced anaemia. A progressive Hb reduction during treatment was observed; however, this reduction was greater in men at week 12 than in women.
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Affiliation(s)
- Nathália Delvaux
- Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Hepatites
Virais, Rio de Janeiro, RJ, Brasil
| | - Vanessa Duarte da Costa
- Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Hepatites
Virais, Rio de Janeiro, RJ, Brasil
| | - Maristella Matos da Costa
- Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Hepatites
Virais, Rio de Janeiro, RJ, Brasil
| | - Livia Melo Villar
- Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Hepatites
Virais, Rio de Janeiro, RJ, Brasil
| | - Henrique Sérgio Moraes Coelho
- Universidade Federal do Rio de Janeiro, Hospital Universitário
Clementino Fraga Filho, Departamento de Hepatologia, Rio de Janeiro, RJ, Brasil
| | - Eliane Bordalo Cathalá Esberard
- Universidade Federal Fluminense, Hospital Universitário Antônio Pedro,
Departamento de Gastroenterologia, Rio de Janeiro, RJ, Brasil
| | - Priscila Pollo Flores
- Universidade Federal Fluminense, Hospital Universitário Antônio Pedro,
Departamento de Gastroenterologia, Rio de Janeiro, RJ, Brasil
| | - Carlos Eduardo Brandão-Mello
- Universidade Federal do Estado do Rio de Janeiro, Hospital Universitário
Gaffrée e Guinle, Departamento de Medicina Geral, Rio de Janeiro, RJ, Brasil
| | - Cristiane Alves Villela-Nogueira
- Universidade Federal do Rio de Janeiro, Hospital Universitário
Clementino Fraga Filho, Departamento de Hepatologia, Rio de Janeiro, RJ, Brasil
| | - Adilson José de Almeida
- Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Hepatites
Virais, Rio de Janeiro, RJ, Brasil
- Universidade Federal do Estado do Rio de Janeiro, Hospital Universitário
Gaffrée e Guinle, Departamento de Medicina Geral, Rio de Janeiro, RJ, Brasil
| | - Elisabeth Lampe
- Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Hepatites
Virais, Rio de Janeiro, RJ, Brasil
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9
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Roberts RL, Barclay ML. Update on thiopurine pharmacogenetics in inflammatory bowel disease. Pharmacogenomics 2015; 16:891-903. [PMID: 26067482 DOI: 10.2217/pgs.15.29] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Azathioprine and 6-mercaptopurine remain pivotal therapies for the maintenance of disease remission in patients with Crohn's disease and ulcerative colitis. While thiopurine S-methyltransferase deficiency was the first pharmacogenetic phenomenon to be recognized to influence thiopurine toxicity and reliably predict leukopenia, it does not predict other adverse effects, nor does it explain most cases of thiopurine resistance. In recent years, a number of other genetic polymorphisms have received increasing attention in the literature. In particular, SNPs in NUDT15 and in the class II HLA locus have been shown to predict thiopurine-related leukopenia and pancreatitis. The aim of this review is to provide a concise update of genetic variability which may influence patient response to azathioprine and 6-mercaptopurine.
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Affiliation(s)
- Rebecca L Roberts
- Department of Surgical Sciences, Dunedin School of Medicine, PO Box 56, Dunedin, New Zealand
| | - Murray L Barclay
- Department of Medicine, University of Otago Christchurch, PO Box 4345, Christchurch, New Zealand.,Department of Gastroenterology, Christchurch Hospital, Private Bag 4710, Christchurch, New Zealand
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10
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Determination of Inosine Triphosphate Pyrophosphatase in Red Blood Cells Using HPLC. Ther Drug Monit 2014; 36:689-91. [DOI: 10.1097/ftd.0000000000000075] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
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Kawaguchi-Suzuki M, Frye RF. The role of pharmacogenetics in the treatment of chronic hepatitis C infection. Pharmacotherapy 2014; 34:185-201. [PMID: 24114761 DOI: 10.1002/phar.1349] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Hepatitis C virus (HCV) chronically infects 170 million people worldwide. Until recently, combination therapy with peginterferon-α (pegIFN) and ribavirin (RBV) has been the standard of care. However, for many patients, especially those infected with the most common HCV genotype 1 (HCV-1), this treatment has resulted in unsatisfactory treatment response rates. Many clinical factors, including pharmacogenetics, influence the treatment response rate. Genetic variation in the interleukin 28B (IL28B) gene is the major determinant of treatment response, a finding that has been replicated in multiple independent cohorts. This review focuses on the association between pharmacogenetics and conventional pegIFN/RBV therapy in patients infected with HCV non-genotype 1; patients reinfected with HCV after liver transplantation; and patients coinfected with HCV and human immunodeficiency virus. We also review the pharmacogenetic data for boceprevir and telaprevir triple therapy in patients with HCV-1 infection, as well as viral genomic polymorphisms and genetic variants that may protect against anemia. Pharmacogenetic information offers a personalized medicine approach to help clinicians and patients make better informed decisions to maximize response and minimize toxicity for the treatment of chronic HCV infection.
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Affiliation(s)
- Marina Kawaguchi-Suzuki
- Department of Pharmacotherapy and Translational Research, College of Pharmacy, University of Florida, Gainesville, Florida
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Simone PD, Pavlov YI, Borgstahl GEO. ITPA (inosine triphosphate pyrophosphatase): from surveillance of nucleotide pools to human disease and pharmacogenetics. Mutat Res 2013; 753:131-146. [PMID: 23969025 DOI: 10.1016/j.mrrev.2013.08.001] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2013] [Revised: 07/31/2013] [Accepted: 08/02/2013] [Indexed: 01/08/2023]
Abstract
Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form and pyrophosphate. Mutations in the ITPA orthologs in model organisms lead to genetic instability and, in mice, to severe developmental anomalies. In humans there is genetic polymorphism in ITPA. One allele leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA deficiency in tissues and plays a role in patients' response to drugs. Structural analysis of this mutant protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active enzyme monomer targets both homo- and heterodimers to degradation.
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Affiliation(s)
- Peter D Simone
- The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Youri I Pavlov
- The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA; Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, USA; Department of Pathology and Microbiology, University of Nebraska Medical Center, USA; Department of Genetics, St-Petersburg University, St-Petersburg, 199034, Russia
| | - Gloria E O Borgstahl
- The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA; Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, USA; Department of Pharmaceutical Sciences, University of Nebraska Medical Center, USA.
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13
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Rau M, Stickel F, Russmann S, Manser CN, Becker PP, Weisskopf M, Schmitt J, Dill MT, Dufour JF, Moradpour D, Semela D, Müllhaupt B, Geier A. Impact of genetic SLC28 transporter and ITPA variants on ribavirin serum level, hemoglobin drop and therapeutic response in patients with HCV infection. J Hepatol 2013. [PMID: 23195617 DOI: 10.1016/j.jhep.2012.11.027] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/09/2023]
Abstract
BACKGROUND & AIMS In the last decade, pegylated interferon-α (PegIFN-α) plus ribavirin (RBV) was the standard treatment of chronic hepatitis C for genotype 1, and it remains the standard for genotypes 2 and 3. Recent studies reported associations between RBV-induced anemia and genetic polymorphisms of concentrative nucleoside transporters such as CNT3 (encoded by SLC28A3) and inosine triphosphatase (encoded by ITPA). We aimed at studying genetic determinants of RBV kinetics, efficacy and treatment-associated anemia. METHODS We included 216 patients from two Swiss study cohorts (61% HCV genotype 1, 39% genotypes 2 or 3). Patients were analyzed for SLC28A2 single nucleotide polymorphism (SNP) rs11854484, SLC28A3 rs56350726, and SLC28A3 rs10868138 as well as ITPA SNPs rs1127354 and rs7270101, and followed for treatment-associated hemoglobin changes and sustained virological response (SVR). In 67 patients, RBV serum levels were additionally measured during treatment. RESULTS Patients with SLC28A2 rs11854484 genotype TT had higher dosage- and body weight-adjusted RBV levels than those with genotypes TC or CC (p=0.02 and p=0.06 at weeks 4 and 8, respectively). ITPA SNP rs1127354 was associated with hemoglobin drop ≥3 g/dl during treatment, in genotype (relative risk (RR)=2.1, 95% CI 1.3-3.5) as well as allelic analyses (RR=2.0, 95%CI 1.2-3.4). SLC28A3 rs56350726 was associated with SVR in genotype (RR=2.2; 95% CI 1.1-4.3) as well as allelic analyses (RR=2.0, 95% CI 1.1-3.4). CONCLUSIONS The newly identified association between RBV serum levels and SLC28A2 rs11854484 genotype, as well as the replicated association of ITPA and SLC28A3 genetic polymorphisms with RBV-induced anemia and treatment response, may support individualized treatment of chronic hepatitis C and warrant further investigation in larger studies.
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Affiliation(s)
- Monika Rau
- Department of Gastroenterology and Hepatology, University Hospital Zurich, Zurich, Switzerland
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the Ochanomizu-Liver Conference Study Group, Nakagawa M, Sakamoto N, Watanabe T, Nishimura-Sakurai Y, Onozuka I, Azuma S, Kakinuma S, Nitta S, Kiyohashi K, Kusano-Kitazume A, Murakawa M, Yoshino K, Itsui Y, Tanaka Y, Mizokami M, Watanabe M. Association of ITPA gene variation and serum ribavirin concentration with a decline in blood cell concentrations during pegylated interferon-alpha plus ribavirin therapy for chronic hepatitis C. Hepatol Int 2013; 7:153-161. [DOI: 10.1007/s12072-012-9363-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
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Tsubota A, Shimada N, Abe H, Yoshizawa K, Agata R, Yumoto Y, Ika M, Namiki Y, Nagatsuma K, Matsudaira H, Fujise K, Tada N, Aizawa Y. Several factors including ITPA polymorphism influence ribavirin-induced anemia in chronic hepatitis C. World J Gastroenterol 2012; 18:5879-5888. [PMID: 23139603 PMCID: PMC3491594 DOI: 10.3748/wjg.v18.i41.5879] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/08/2012] [Revised: 05/28/2012] [Accepted: 06/28/2012] [Indexed: 02/06/2023] Open
Abstract
AIM To construct formulae for predicting the likelihood of ribavirin-induced anemia in pegylated interferon α plus ribavirin for chronic hepatitis C. METHODS Five hundred and sixty-one Japanese patients with hepatitis C virus genotype 1b who had received combination treatment were enrolled and assigned randomly to the derivation and confirmatory groups. Single nucleotide polymorphisms at or nearby ITPA were genotyped by real-time detection polymerase chain reaction. Factors influencing significant anemia (hemoglobin concentration < 10.0 g/dL at week 4 of treatment) and significant hemoglobin decline (declining concentrations > 3.0 g/dL at week 4) were analyzed using multiple regression analyses. Prediction formulae were constructed by significantly independent factors. RESULTS Multivariate analysis for the derivation group identified four independent factors associated with significant hemoglobin decline: hemoglobin decline at week 2 [P = 3.29 × 10(-17), odds ratio (OR) = 7.54 (g/dL)], estimated glomerular filtration rate [P = 2.16 × 10(-4), OR = 0.962 (mL/min/1.73 m(2))], rs1127354 (P = 5.75 × 10(-4), OR = 10.94) and baseline hemoglobin [P = 7.86 × 10(-4), OR = 1.50 (g/dL)]. Using the model constructed by these factors, positive and negative predictive values and predictive accuracy were 79.8%, 88.8% and 86.2%, respectively. For the confirmatory group, they were 83.3%, 91.0% and 88.3%. These factors were closely correlated with significant anemia. However, the model could not be constructed, because no patients with rs1127354 minor genotype CA/AA had significant anemia. CONCLUSION Reliable formulae for predicting the likelihood of ribavirin-induced anemia were constructed. Such modeling may be useful in developing individual tailoring and optimization of ribavirin dosage.
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Bierau J, Bakker JA, Schippers JA, Grashorn JAC, Lindhout M, Lowe SH, Paulussen ADC, Verbon A. Erythrocyte inosine triphosphatase activity is decreased in HIV-seropositive individuals. PLoS One 2012; 7:e30175. [PMID: 22272297 PMCID: PMC3260219 DOI: 10.1371/journal.pone.0030175] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2011] [Accepted: 12/13/2011] [Indexed: 01/28/2023] Open
Abstract
Background Inosine triphosphatase (ITPase) is encoded by the polymorphic gene ITPA and maintains low intracellular levels of the inosine nucleotides ITP and dITP. The most frequently reported polymorphisms are ITPA c.94C>A (rs 1127354) and ITPA c. 124+21 A>C (rs7270101). Some nucleoside-analogues used in the treatment of HIV-seropositive (HIV+) patients are potential substrates for ITPase. Therefore, the frequency of ITPA SNPs and ITPase activity were studied in a population of HIV+-patients. Methods The study population consisted of 222 patients, predominantly Caucasian males, >95% using HAART. Erythrocyte ITPase activity was determined by measuring the formation of IMP from ITP. ITPA genotype was determined by sequencing genomic DNA. Distribution of ITPase activity, genotype-phenotype correlation and allele frequencies were compared to 198 control subjects. The effect of nucleoside analogues on ITPase activity was studied using lymphoblastic T-cell cultures and human recombinant ITPase. Enzyme kinetic experiments were performed on erythrocyte ITPase from HIV+ patients and controls. Results No difference was observed in the allele frequencies between the HIV+-cohort (± HAART) and the control population. HIV+ carriers of the wild type and ITPA c.94C>A had significantly lower ITPase activities than control subjects with the same genotype (p<0.005). This was not observed in ITPA c. 124+21 A>C carriers. Nucleoside analogues did not affect ITPase activity in cell culture and human recombinant ITPase. Conclusion: ITPA population genetics were identical in HIV+ and control populations. However, the majority of HIV+-patients had decreased erythrocyte ITPase activity compared to controls, probably due to decreased amounts of ITPase protein. It seems unlikely that ITPase activity is decreased due to nucleoside analogues (HAART). Long-term effects of HIV-infection altering ITPase protein expression or stability may explain the phenomenon observed.
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Affiliation(s)
- Jörgen Bierau
- Department of Clinical Genetics, Maastricht University Medical Centre, Maastricht, The Netherlands.
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Tanaka Y, Kurosaki M, Nishida N, Sugiyama M, Matsuura K, Sakamoto N, Enomoto N, Yatsuhashi H, Nishiguchi S, Hino K, Hige S, Itoh Y, Tanaka E, Mochida S, Honda M, Hiasa Y, Koike A, Sugauchi F, Kaneko S, Izumi N, Tokunaga K, Mizokami M. Genome-wide association study identified ITPA/DDRGK1 variants reflecting thrombocytopenia in pegylated interferon and ribavirin therapy for chronic hepatitis C. Hum Mol Genet 2011; 20:3507-3516. [PMID: 21659334 DOI: 10.1093/hmg/ddr249] [Citation(s) in RCA: 72] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Hematologic abnormalities during current therapy with pegylated interferon and ribavirin (PEG-IFN/RBV) for chronic hepatitis C (CHC) often necessitate dose reduction and premature withdrawal from therapy. The aim of this study was to identify host factors associated with IFN-induced thrombocytopenia by genome-wide association study (GWAS). In the GWAS stage using 900K single-nucleotide polymorphism (SNP) microarrays, 303 Japanese CHC patients treated with PEG-IFN/RBV therapy were genotyped. One SNP (rs11697186) located on DDRGK1 gene on chromosome 20 showed strong associations in the minor-allele-dominant model with the decrease of platelet counts in response to PEG-IFN/RBV therapy [P = 8.17 × 10(-9); odds ratio (OR) = 4.6]. These associations were replicated in another sample set (n = 391) and the combined P-values reached 5.29 × 10(-17) (OR = 4.5). Fine mapping with 22 SNPs around DDRGK1 and ITPA genes showed that rs11697186 at the GWAS stage had a strong linkage disequilibrium with rs1127354, known as a functional variant in the ITPA gene. The ITPA-AA/CA genotype was independently associated with a higher degree of reduction in platelet counts at week 4 (P < 0.0001), as well as protection against the reduction in hemoglobin, whereas the CC genotype had significantly less reduction in the mean platelet counts compared with the AA/CA genotype (P < 0.0001 for weeks 2, 4, 8, 12), due to a reactive increase of the platelet count through weeks 1-4. Our present results may provide a valuable pharmacogenetic diagnostic tool for tailoring PEG-IFN/RBV dosing to minimize drug-induced adverse events.
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Affiliation(s)
- Yasuhito Tanaka
- Department of Virology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
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Cariani E, Villa E, Rota C, Critelli R, Trenti T. Translating pharmacogenetics into clinical practice: interleukin (IL)28B and inosine triphosphatase (ITPA) polymophisms in hepatitis C virus (HCV) infection. Clin Chem Lab Med 2011; 49:1247-1256. [PMID: 21612542 DOI: 10.1515/cclm.2011.618] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Hepatitis C virus (HCV) infection is frequently characterized by evolution to chronicity and by a variable clinical course of the disease. The clinical heterogeneities of HCV infection and the imperfect predictability of the response to interferon (IFN) have suggested the need to search for a genetic basis of clinical features. This led to the discovery of genetic polymorphisms playing a major role in the evolution of infection, as well as on treatment response and adverse effects. This review will cover recent reports on the subject, focusing on the potential use of the new genetic markers in the diagnostic algorithm for the stratification of patients for personalized antiviral regimens.
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Affiliation(s)
- Elisabetta Cariani
- Clinical Pathology-Toxicology, Ospedale Civile S. Agostino-Estense, Modena, Italy
| | - Erica Villa
- Department of Gastroenterology, Azienda Ospedaliera-Universitaria, University of Modena and Reggio Emilia, Modena, Italy
| | - Cristina Rota
- Clinical Pathology-Toxicology, Ospedale Civile S. Agostino-Estense, Modena, Italy
| | - Rosina Critelli
- Department of Gastroenterology, Azienda Ospedaliera-Universitaria, University of Modena and Reggio Emilia, Modena, Italy
| | - Tommaso Trenti
- Clinical Pathology-Toxicology, Ospedale Civile S. Agostino-Estense, Modena, Italy
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Sakamoto N, Tanaka Y, Nakagawa M, Yatsuhashi H, Nishiguchi S, Enomoto N, Azuma S, Nishimura-Sakurai Y, Kakinuma S, Nishida N, Tokunaga K, Honda M, Ito K, Mizokami M, Watanabe M. ITPA gene variant protects against anemia induced by pegylated interferon-α and ribavirin therapy for Japanese patients with chronic hepatitis C. Hepatol Res 2010; 40:1063-1071. [PMID: 20977565 DOI: 10.1111/j.1872-034x.2010.00741.x] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
AIM Host genetic variants leading to inosine triphosphatase (ITPA) deficiency, a condition not thought to be clinically important, protect against hemolytic anemia in chronic hepatitis C patients receiving ribavirin. In this study, we evaluated the clinical significance of ITPA variants in Japanese hepatitis C patients who were treated with pegylated interferon plus ribavirin. METHODS In this multicenter retrospective cross-sectional study, 474 hepatitis C patients were enrolled who were treated with pegylated interferon plus ribavirin in four geographically different hospitals in Japan. Patients were grouped according to hemoglobin decline of more than 3 g/dL at week 4. Two single nucleotide polymorphisms (SNP) within or adjacent to the ITPA gene (rs6051702, rs1127354) were genotyped. RESULTS A functional SNP, rs1127354, within the ITPA exon was strongly associated with protection against anemia with only one (0.8%) in 129 patients with the ITPA minor variant A developing severe anemia (P=5.9×10(-20) ). For rs6051702, which had significant association in European-Americans, significant but weak association with severe hemoglobin reduction was found in Japanese (P= 0.009). In patients excluding genotype 1b and high viral load, those with the ITPA minor variant A achieved significantly higher sustained viral response rate than those with the major variant (CC) (96% vs 70%, respectively, P= 0.0066). CONCLUSION ITPA SNP, rs1127354, is confirmed to be a useful predictor of ribavirin-induced anemia in Japanese patients. Patients with the ITPA minor variant A (~ 27%) have an advantage in pegylated interferon plus ribavirin-based therapies, due to expected adherence of ribavirin doses, resulting in a higher viral clearance rate.
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Affiliation(s)
- Naoya Sakamoto
- Department of Gastroenterology and Hepatology Department for Hepatitis Control, Tokyo Medical and Dental University, Tokyo, Japan
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Fellay J, Thompson AJ, Ge D, Gumbs CE, Urban TJ, Shianna KV, Little LD, Qiu P, Bertelsen AH, Watson M, Warner A, Muir AJ, Brass C, Albrecht J, Sulkowski M, McHutchison JG, Goldstein DB. ITPA gene variants protect against anaemia in patients treated for chronic hepatitis C. Nature 2010; 464:405-8. [PMID: 20173735 DOI: 10.1038/nature08825] [Citation(s) in RCA: 370] [Impact Index Per Article: 24.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2009] [Accepted: 01/11/2010] [Indexed: 12/15/2022]
Abstract
Chronic infection with the hepatitis C virus (HCV) affects 170 million people worldwide and is an important cause of liver-related morbidity and mortality. The standard of care therapy combines pegylated interferon (pegIFN) alpha and ribavirin (RBV), and is associated with a range of treatment-limiting adverse effects. One of the most important of these is RBV-induced haemolytic anaemia, which affects most patients and is severe enough to require dose modification in up to 15% of patients. Here we show that genetic variants leading to inosine triphosphatase deficiency, a condition not thought to be clinically important, protect against haemolytic anaemia in hepatitis-C-infected patients receiving RBV.
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Affiliation(s)
- Jacques Fellay
- Institute for Genome Sciences & Policy, Center for Human Genome Variation, Duke University, Durham, North Carolina 27708, USA
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Roberts RL, Gearry RB, Kennedy MA, Barclay ML. Beyond TPMT: genetic influences on thiopurine drug responses in inflammatory bowel disease. Per Med 2008; 5:233-248. [PMID: 29783500 DOI: 10.2217/17410541.5.3.233] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Azathioprine and 6-mercaptopurine are widely used in the management of inflammatory bowel disease (IBD). However, approximately 25% of IBD patients experience toxicity, and up to 10% show resistance to these thiopurine drugs. The importance of genetic variability in determining thiopurine toxicity was first recognized over 25 years ago with the discovery of the thiopurine S-methyltransferase (TPMT) polymorphism and the occurrence of azathioprine-induced myelosuppression in TPMT-deficient patients. In the intervening period, TPMT has become the foremost example of pharmacogenetics, and TPMT deficiency represents one of the few pharmacogenetic phenomena that have successfully made the transition from the research laboratory to diagnostics. While TPMT activity predicts some cases of myelosuppression, deficiency in this enzyme is neither predictive of other adverse drug reactions, nor resistance to thiopurine therapy. As myelosuppression only accounts for approximately 2.5% of adverse reactions in IBD patients, researchers are increasingly turning their attention to other enzymes involved in thiopurine metabolism to find molecular explanations for intolerance and resistance to azathioprine and 6-mercaptopurine. In this review, we summarize the current state of knowledge with regards to TPMT, and also explore genetic variability, beyond TPMT, that may contribute to thiopurine response in IBD patients.
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Affiliation(s)
| | - Richard B Gearry
- Department of Medicine, University of Otago, Christchurch 8140, New Zealand.,Department of Gastroenterology, Christchurch Hospital, Private Bag 151, Christchurch 8140, New Zealand
| | - Martin A Kennedy
- Department of Pathology, University of Otago, Christchurch 8140, New Zealand
| | - Murray L Barclay
- Department of Medicine, University of Otago, Christchurch 8140, New Zealand.,Department of Gastroenterology, Christchurch Hospital, Private Bag 151, Christchurch 8140, New Zealand
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von Ahsen N, Oellerich M, Armstrong VW. Characterization of the inosine triphosphatase (ITPA) gene: haplotype structure, haplotype-phenotype correlation and promoter function. Ther Drug Monit 2008; 30:16-22. [PMID: 18223458 DOI: 10.1097/ftd.0b013e318161a21a] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
Inosine triphosphatase (ITPA) cleaves phosphate residues from inosine triphosphate (ITP) and deoxy ITP (dITP), thereby recovering inosine monophosphate, which is a substrate for further purine nucleotide pathways. Deficient ITPA activity leads to intracellular accumulation of ITP/dITP and would, under thiopurine therapy, lead to accumulation of unusual thio-inosine metabolites (thio-ITP) with the potential for adverse metabolic effects. ITPA is a promising candidate for a more comprehensive understanding of thiopurine pharmacogenetics. We therefore studied the haplotype structure, haplotype-phenotype association, and promoter function of ITPA in a Western European population.ITPA haplotyping was performed based on haplotype tagging SNPs (selected from HapMap data) in healthy 130 controls. Haplotypes were reconstructed, and the haplotype-phenotype association was assessed by haplotype trend regression. A 1.5 kb upstream region and stepwise deletions thereof were tested for promoter activity in reporter gene assays in HepG2 and CCRF-CEM cells. Transcription factor binding (Sp1, Sp3) to the proximal promoter region was studied by chromatin immunoprecipitation. Among haplotypes with a frequency greater than 0.01, we did not find any new low-activity haplotypes besides those carrying 94C>A or IVS2 + 21A>C variants. Two promoter SNPs had no influence on promoter activity. An approximately 200 bp sized GC-rich proximal promoter region was sufficient to fully drive reporter gene activity. Chromatin immunoprecipitation showed binding of Sp1 and Sp3 transcription factors to this region. Only the two haplotypes carrying 94C>A or IVS2 + 21A>C were associated with reduced enzyme activity. The gene promoter is associated with a CpG island and driven by Sp-family transcription factors. There was no evidence for functional promoter SNPs, and it is suggested that only SNPs within the very proximal promoter region (approximately 200 bp) have the potential to be functionally significant.
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Abstract
Inosine triphosphatase (ITPase) is the enzyme that catalyzes the conversion of inosine triphosphate (ITP) and deoxy-inosine triphosphate (dITP) to inosine monophosphate and deoxy-inosine monophosphate, respectively, thereby maintaining low intracellular concentrations of ITP and dITP. Individuals deficient in ITPase activity were first recognized over 30 years ago. For decades, no clinical significance could be attributed to this inborn error of metabolism whatsoever. In recent years, evidence has started to accumulate that polymorphisms in the gene encoding ITPase are associated with potentially severe adverse drug reactions towards the thiopurine drugs azathioprine and 6-mercaptopurine. The pharmacogenetic significance is presently being debated in the literature. However, the present state of knowledge is still insufficient to definitively determine the pharmacogenetic significance of ITPase. This article aims to review the current knowledge on the role of ITPase in thiopurine metabolism.
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Affiliation(s)
- Jörgen Bierau
- Maastricht University Hospital, Laboratory of Biochemical Genetics, Department of Clinical Genetics, PO Box 6202 AZ Maastricht, The Netherlands.
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ZHENG QINGCHUAN, SUN CHIACHUNG. HOMOLOGY MODELING AND MOLECULAR DYNAMICS STUDY OF HUMAN INOSINE TRIPHOSPHATE PYROPHOSPHATASE. JOURNAL OF THEORETICAL & COMPUTATIONAL CHEMISTRY 2007. [DOI: 10.1142/s0219633607002824] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
With homology-modeling techniques, molecular mechanics and molecular dynamics methods, a 3D structure model of the human inosine triphosphatase (ITPase; EC 3.6.1.19) is created and refined. This model is further assessed by Profile-3D and ProStat, which confirm that the refined model is reliable. With this model, a flexible docking study is performed, and the results indicate that Arg178, Lys19 and Glu44 are three important determinant residues in substrate binding because they have prominent interaction energies with ITP and form strong hydrogen bonds with ITP. In addition, we further find that the P32T substitution alters the α-helices of ITPase but the β-sheets are almost not changed, and the mutation induces the interaction energy between ITPase and ITP to increase, which are consistent with the conclusion predicted by Sumi et al.8 The results from the mutagenesis imply that Pro32 is vital for the catalytic activity.
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Affiliation(s)
- QING-CHUAN ZHENG
- State Key Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun 130023, P. R. China
| | - CHIA-CHUNG SUN
- State Key Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun 130023, P. R. China
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25
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Atanasova S, Shipkova M, Svinarov D, Mladenova A, Genova M, Wieland E, Oellerich M, von Ahsen N. Analysis of ITPA phenotype-genotype correlation in the Bulgarian population revealed a novel gene variant in exon 6. Ther Drug Monit 2007; 29:6-10. [PMID: 17304144 DOI: 10.1097/ftd.0b013e3180308554] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Mutations in the inosine triphosphate pyrophosphohydrolase (ITPA) gene causing enzyme deficiency were shown to have pharmacogenetic implications in azathioprine-induced adverse drug reactions. The distribution of ITPA activity as well as the types and the frequencies of gene variants associated with a lower enzyme activity were determined in healthy volunteers from a Bulgarian population. The ITPA activity was measured in 185 erythrocyte samples by an established high-performance liquid chromatography procedure. All samples were genotyped for 94C > A, IVS2 + 21A > C, and IVS2 + 68T > C/G by real-time polymerase chain reaction with hybridization probes. The ITPA activity ranged from 7.5 to 587.8 micromoL IMP/(g Hb x h) with a median value of 162.9 micromoL IMP/(g Hb x h). The enzyme activity showed significant differences between females and males (P = 0.006) with 17% higher values in men than women. Mutant allele frequencies were 0.038 (94C > A) and 0.130 (IVS2 + 21A > C). Mutations at IVS2 + 68 were not identified. Using a cutoff at 75 micromoL IMP/(g Hb x h) phenotyping detected all heterozygous carriers of 94C > A, two compound heterozygotes, all IVS2 + 21A > C homozygotes and 12.5% of IVS2 + 21A > C heterozygous cases. A novel frameshift mutation 359_366dupTCAGCACC in exon 6 was found in a subject with reduced enzyme activity of 61.2 micromoL IMP/(g Hb x h). The interindividual variability in ITPA activity among the Bulgarian population resembles the distribution of enzyme activity in other whites, although the observed median activity was approximately 25% lower in the Bulgarians [163 vs 219 micromoL IMP/(g Hb x h)]. The most common mutant allele IVS2 + 21A > C showed a similar frequency like in other white populations, whereas the 94C > A mutation was less frequently observed compared with other whites. Heterozygosity for the novel gene variant 359_366dupTCAGCACC was associated with 30% enzyme activity of the wild-type median value. The role of this rare variant for the thiopurine intolerance is not explored. These data further extend the knowledge for ITPA heterogeneity in whites.
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Affiliation(s)
- Srebrena Atanasova
- Department of Clinical Chemistry, Georg-August University Goettingen, Goettingen, Germany.
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Arenas M, Duley J, Sumi S, Sanderson J, Marinaki A. The ITPA c.94C>A and g.IVS2+21A>C sequence variants contribute to missplicing of the ITPA gene. Biochim Biophys Acta Mol Basis Dis 2006; 1772:96-102. [PMID: 17113761 DOI: 10.1016/j.bbadis.2006.10.006] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2006] [Revised: 09/18/2006] [Accepted: 10/02/2006] [Indexed: 12/14/2022]
Abstract
Inosine triphosphate pyrophosphatase (ITPase) catalyzes the conversion of inosine triphosphate (ITP) to the correspondent monophosphate. The ITPA c.94C>A and g.IVS2+21A>C allelic variants are associated with decreased red cell enzyme activity. The ITPA c.94C>A [P32T] sequence variant is associated with an increased risk of adverse drug reactions in patients treated with the thiopurine drug azathioprine. The aim of this study was to explore the molecular mechanisms of ITPase deficiency. ITPA mRNA was extracted from peripheral blood leukocytes (PBL), Epstein-Barr virus transformed lymphoblast cell cultures, reticulocytes, and cultured fibroblast from patients with known ITPA genotypes. ITPA mRNA was reversed transcribed, sequenced and the relative amounts of misspliced transcripts quantitated from three independent experiments. The ITPA g.IVS2+21A>C sequence variant resulted in missplicing of exon 3. The ITPA c.94C>A allelic variant resulted in missplicing of exons 2 and 3 representing, in PBL samples, 61% of the total mRNA expressed in ITPA c.94C>A homozygotes. We proposed that the ITPA c.94C>A allelic variant destroys an exonic splicing silencing (ESS) element in exon 2, resulting in the activation of two nearby upstream 5' splice sites and missplicing of the exons 2 and 3 cassette causing structural changes to the enzyme and contributing to ITPase deficiency.
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Affiliation(s)
- Monica Arenas
- Purine Research Laboratory, Department of Chemical Pathology, Guy's Hospital, London, SE1 9RT, UK, and Department of Paediatrics, Neonatology and Congenital Disorders, Nagoya City University, Japan
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Abstract
BACKGROUND In the past 10-20 years, knowledge of both thiopurine pharmacology and -pharmacogenetics has been extended dramatically and used to develop new strategies to improve efficacy and reduce toxicity. AIM To review thiopurine efficacy, toxicity, pharmacology, pharmacogenetics, interactions in patients with inflammatory bowel disease. Special attention was paid to new strategies for optimization of pharmacotherapy. METHODS To collect relevant scientific articles, a Pubmed search was performed from 1966 through January 2006 with the following key words (MeSH terms preferentially) in multiple combinations: 'azathioprine', '6-mercaptopurine', '6-MP', '6-thioguanine', '6-TG', 'thiopurine(s)', 'metabolites', 'level(s)', 'TDM', 'TMPT', 'ITPA', 'genotype(s)', 'phenotype(s)', 'inflammatory bowel disease', 'Crohn('s) disease', 'ulcerative colitis'. RESULTS Strategies for optimization of pharmacotherapy include therapeutic drug monitoring of thiopurine metabolites, geno- or phenotyping crucial enzymes in thiopurine metabolism like thiopurine S-methyltransferase and inosine triphosphate pyrophosphatase, and the use of thioguanine as such. CONCLUSIONS Thiopurine S-methyltransferase genotyping and therapeutic drug monitoring are useful instruments for individualizing thiopurine pharmacotherapy of inflammatory bowel disease. Inosine triphosphate pyrophosphatase genotyping may be helpful in case of unexplainable myelotoxicity. In case of azathioprine- or mercaptopurine-intolerance, thioguanine seems a promising alternative. However, more knowledge needs to be gathered about its potential hepatotoxicity.
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Affiliation(s)
- L J J Derijks
- Department of Clinical Pharmacy, Máxima Medical Center, Veldhoven, The Netherlands.
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28
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Shipkova M, Lorenz K, Oellerich M, Wieland E, von Ahsen N. Measurement of erythrocyte inosine triphosphate pyrophosphohydrolase (ITPA) activity by HPLC and correlation of ITPA genotype-phenotype in a Caucasian population. Clin Chem 2006; 52:240-7. [PMID: 16384889 DOI: 10.1373/clinchem.2005.059501] [Citation(s) in RCA: 85] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
BACKGROUND Inosine triphosphate (ITP) pyrophosphohydrolase (ITPA) catalyzes the pyrophosphohydrolysis of ITP/dITP and xanthosine triphosphate to prevent incorporation of unusual nucleotides into RNA and DNA. Important mutations leading to enzyme deficiency are 94C>A and IVS2 + 21A>C. An association between ITPA 94C>A and adverse reactions during azathioprine treatment has been shown. To investigate the ITPA phenotype, an HPLC procedure was developed and phenotype-genotype correlations were assessed. METHODS The enzymatic conversion of ITP to inosine monophosphate (IMP) was terminated by perchloric acid and saturated dipotassium hydrogen phosphate. We quantified the IMP at 262 nm after separation on an Aqua perfect C18 column using 20 mmol/L phosphate buffer, pH 2.5. We also genotyped samples for ITPA 94C>A and IVS2 + 21A>C by real-time fluorescence PCR. RESULTS The assay was linear to 3 mmol/L IMP [approximately 500 micromol/(g Hb x h)] with a lower limit of quantification of 4 micromol/L [approximately 0.5 micromol/(g Hb x h)]. With IMP-enriched samples, within- and between-day imprecision was < or = 3.6% and < or = 4.9%, respectively, and the inaccuracy was < or = 5.2%. With pooled erythrocytes, within- and between-day imprecision was 3.8% and 7.5%, respectively. ITPA activity in 130 healthy controls was between < 0.5 and 408 micromol IMP/(g Hb x h). Mutant allele frequencies were 0.062 (94C>A) and 0.131 (IVS2 + 21A>C). When we used a cutoff of 125 micromol IMP/(g Hb x h), phenotyping detected all 94C>A mutant cases, all 94C>A and IVS2 + 21A>C compound heterozygotes, all IVS2 + 21A>C homozygotes, and 6 of 24 IVS2 + 21A>C heterozygote-only cases. A novel IVS2 + 68T>C mutation was also found. CONCLUSIONS The HPLC procedure provides an excellent ITPA phenotype-genotype correlation and led to the discovery of a novel IVS2 + 68T>C mutation. The method could facilitate investigation of the role of ITPA activity for drug toxicity during thiopurine therapy.
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Affiliation(s)
- Maria Shipkova
- Central Institute of Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Stuttgart, Germany.
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Abstract
Inherited variations in the nucleotide sequence of genes influence how individual patients respond to drugs. Most commonly, clinically significant genetic variations consist of single nucleotide polymorphisms (SNPs) within genes that affect drug disposition or drug targets. Up to now, relatively few clinically important examples of inherited traits that affect drug responses have been studied in detail. However, one of the well-characterized examples is highly relevant to inflammatory bowel disease therapeutics, that of thiopurine methyltransferase pharmacogenetics. Individuals with 2 normal alleles of the gene encoding thiopurine methyltransferase metabolize and clear thiopurines such as azathioprine and 6-mercaptopurine rapidly. Individuals with 1 normal and 1 variant allele are intermediate, whereas those with 2 variant alleles clear thiopurines very slowly. Intermediate and slow metabolizers are predisposed to have high active thiopurine drug levels and develop bone marrow suppression. Genomic era technology permits determination of large numbers of SNPs in large numbers of individuals. This capability is allowing the field of pharmacogenomics to become one of the most productive interfaces in translational biomedical research at present. By using high-throughput SNP genotyping, combined with careful phenotypic characterization of disease, pharmacogenomic research carries the potential of identifying individual biomarkers that predict the relative likelihood of benefit or risk from a therapeutic intervention. If this promise can be realized, pharmacogenomics will deliver the opportunity for personalized medicine.
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Affiliation(s)
- Laurence J Egan
- Department of Pharmacology, National University of Ireland, Galway, Ireland.
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Maeda T, Sumi S, Ueta A, Ohkubo Y, Ito T, Marinaki AM, Kurono Y, Hasegawa S, Togari H. Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency in the Japanese population. Mol Genet Metab 2005; 85:271-9. [PMID: 15946879 DOI: 10.1016/j.ymgme.2005.03.011] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/26/2004] [Revised: 03/24/2005] [Accepted: 03/26/2005] [Indexed: 11/29/2022]
Abstract
Inosine triphosphate pyrophosphohydrolase (ITPase) is an enzyme that catalyzes the conversion of inosine triphosphate (ITP) to inosine monophosphate and pyrophosphate. In Caucasian populations it is reported that the frequency of cases showing decreased ITPase activity is 5%. The structure of ITPA gene along with five single nucleotide polymorphisms has been reported in Caucasians. We examined ITPase activity and frequency of two polymorphisms (94C>A and IVS2+21A>C) in 100 Japanese individuals. Among these individuals, we observed that three cases with zero activity were homozygote for 94C>A, and were accompanied by abnormal accumulation of ITP in erythrocytes. The cases included in the low ITPase activity group were heterozygote for 94C>A polymorphism. The activity of the heterozygote cases was approximately 27% of the mean value of the wild type. The allele frequency of the 94C>A polymorphism was 0.155, which was 2.6 times higher than that of the Caucasians (0.06). The IVS2+21A>C was not detected in Japanese cases, although it occurred with a frequency of 0.130 in Caucasians. Furthermore, we identified a novel mutation IVS2+68T>G in intron 2 in the case with the lowest enzyme activity in the 94C>A wild type. Since the frequency of ITPA 94C>A polymorphism is higher in the Japanese population than that in Caucasians, it is more important to examine ITPA 94C>A polymorphism in the Japanese population to prevent thiopurine drug toxicity. Pretherapeutic screening of individuals for ITPA polymorphisms should be considered for safer and more tolerable treatment with thiopurine drugs.
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Affiliation(s)
- Tohru Maeda
- Department of Hospital Pharmacy, Nagoya City University Hospital, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8602, Japan.
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31
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Rodriguez DB, Mackin A, Easley R, Boyle CR, Hou W, Langston C, Walsh AM, Province MA, McLeod HL. Relationship between Red Blood Cell Thiopurine Methyltransferase Activity and Myelotoxicity in Dogs Receiving Azathioprine. J Vet Intern Med 2004. [DOI: 10.1111/j.1939-1676.2004.tb02555.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
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Duley JA, Simmonds HA, Hopkinson DA, Levinsky RJ. Inosine triphosphate pyrophosphohydrolase deficiency in a kindred with adenosine deaminase deficiency. Clin Chim Acta 1990; 188:243-52. [PMID: 2167185 DOI: 10.1016/0009-8981(90)90206-8] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
A complete deficiency of inosine triphosphate pyrophosphohydrolase (ITPase) has been identified, together with high concentrations (mean 157 mumol/l) of the unusual nucleotide ITP, in the erythrocytes of 3 members of a consanguineous United Kingdom kindred. The defect has been noted previously in North America and Sweden, but even in presumed homozygotes some residual ITPase activity was reported. Homozygosity for the defect has not been associated previously with any clinical abnormality. In this kindred it was co-existent with adenosine deaminase (ADA) deficient severe combined immunodeficiency. Since the genes for both ITPase and ADA are localised on the same chromosome, segregation analysis of ITPase and ADA activity was undertaken in available kindred members. The results confirmed an autosomal recessive mode of inheritance for ITPase deficiency, but suggested that the co-existence with ADA deficiency was coincidental.
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Affiliation(s)
- J A Duley
- Purine Research Laboratory, UMDS Guy's Hospital, London, UK
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33
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Matias C, Nott SE, Bagnara AS, O'Sullivan WJ, Gero AM. Purine salvage and metabolism in Babesia bovis. Parasitol Res 1990; 76:207-13. [PMID: 1690419 DOI: 10.1007/bf00930816] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Studies of the incorporation of radio-labelled purine precursors into the erythrocytic forms of Babesia bovis under tissue-culture conditions have confirmed the presence in the parasite of enzymatic activities responsible for the salvage of preformed purines. The results also revealed that the parasite was capable of a variety of nucleotide interconversions, such that exogenous hypoxanthine and adenosine were incorporated into both adenine and guanine nucleotides followed by the incorporation of these nucleotides into the adenine and guanine moieties of RNA and DNA. No evidence was found for salvage of preformed pyrimidines. Evidence was also obtained for the insertion of a parasite-specific nucleoside/nucleobase transporter into the membrane of the bovine (host) red cell. Thus, whereas normal (non-parasitised) bovine red cells are essentially incapable of transporting nucleosides across their membranes, the invasion of these cells by B. bovis introduces a transporter that can be inhibited by classic nucleoside transport inhibitors.
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Affiliation(s)
- C Matias
- School of Biochemistry, University of New South Wales, Kensington, Australia
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Simmonds HA, Micheli V, Duley JA, Fairbanks LD, Hopkinson DA, Levinsky RJ. Further evidence for a 'new' purine defect, inosine triphosphate (ITP) pyrophosphohydrolase deficiency. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 1989; 253A:97-102. [PMID: 2560342 DOI: 10.1007/978-1-4684-5673-8_15] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Affiliation(s)
- H A Simmonds
- Purine Research Laboratory, UMDS Guy's Hospital, Siena, Italy
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35
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van Waeg G, Niklasson F, Ericson A, de Verdier CH. Purine metabolism in normal and ITP-pyrophosphohydrolase-deficient human erythrocytes. Clin Chim Acta 1988; 171:279-92. [PMID: 2836113 DOI: 10.1016/0009-8981(88)90154-4] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Fresh and stored erythrocytes from normal and ITP-pyrophosphohydrolase (ITP-ase, EC 3.6.1.19) deficient individuals were incubated with hypoxanthine, guanine, allopurinol, and inosine. Differences in the purine metabolism between the normal and the ITP-ase deficient erythrocytes were observed only in the IMP-ITP cycle. Hypoxanthine, guanine and allopurinol were converted to nucleotides at the same rate. Hypoxanthine (2.5 mumol/l) inhibited the salvage of allopurinol (40 mumol/l). A slow decrease (0.7%/day) in salvage rate was observed in both types of cells upon storage at +4 degrees C. Erythrocyte ITP-ase activity was measured in a reference sample group of 48 healthy volunteers. Two distinct groups were found with mean activities equal to 48.3 +/- 13.1 nkat/g Hb (means +/- SD, n = 38) and 11.4 +/- 4.3 nkat/g Hb (n = 10). In two previously selected subjects, the ITP-ase activity was 0.2 and 2.4 nkat/g Hb. A hypothetical genetic mechanism is discussed. The maximal energy turnover in the IMP-ITP cycle during hypoxanthine incubation was found to be less than 10% of the basal erythrocyte energy turnover.
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Affiliation(s)
- G van Waeg
- Departmet of Clinical Chemistry, Uppsala University, Akademiska Sjukhuset, Sweden
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Hallam LJ, Van der Weyden MB, Ackland SP, Bagnara AS, Whiteside MG. The biochemical and clinical consequences of 2'-deoxycoformycin in T cell chronic lymphocytic leukaemia. SCANDINAVIAN JOURNAL OF HAEMATOLOGY 1984; 32:55-64. [PMID: 6607510 DOI: 10.1111/j.1600-0609.1984.tb00678.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
The mechanisms for cell toxicity with adenosine deaminase inhibition by 2'-deoxycoformycin (dCF) in non replicating lymphoid cells include S-adenosylhomocysteine (SAH) hydrolase inactivation and reduction of cellular ATP content. These postulates were explored in a patient with T-CLL receiving dCF with a resultant fall in peripheral blood lymphocytes from 740 X 10(9)/1 to 90 X 10(9)/1 over 15 d. In red cells there was complete inhibition of adenosine deaminase and SAH hydrolase activities, progressive deoxyadenosine triphosphate (dATP) accumulation and ATP depletion but no significant alteration in adenosine monophosphate (AMP) deaminase activity or distribution in purine intermediates from radioactive adenosine. In T-CLL lymphocytes, there was incomplete lymphoid SAH hydrolase inactivation, reduced AMP deaminase activity and progressive dATP accumulation. The limited decrease in lymphocyte ATP content was related more to dCF administration than dATP accumulation, nor accompanied by significant changes in the distribution of purine intermediates from adenosine. These findings suggest that ATP depletion with dCF therapy does not reflect AMP deaminase activity modulation nor is of critical importance for cell toxicity. The exact role for elevated cellular dATP content and SAH hydrolase inactivation in this toxicity remains to be established.
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Verhoef VL, Fuller SA, Morris AJ. Individual variation of nucleoside triphosphate pyrophosphohydrolase activity in human erythrocytes, granulocytes, lymphocytes, and platelets. Biochem Genet 1980; 18:235-45. [PMID: 6108768 DOI: 10.1007/bf00484239] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Analysis of the nucleoside triphosphate pyrophosphohydrolase specific activity of red cells obtained from a random Caucasian population indicated at least two subclasses. The specific activity of 18% of the population ranged from undetectable activity to 27.5 nmol ITP cleaved/20 min/mg hemoglobin. The remainder of the population had higher activity, 27.5-125 nmoles ITP cleaved/20 min/mg hemoglobin. The variation of NTPH activity evident in the red cells of an individual is reflected in granulocytes, lymphocytes, and platelets of that individual. Erythrocyte activity ranges from 0.7 to 21 units (nmol of ITP cleaved in 20 min)/10(7) cells, granulocytes have 17-201 units/10(7) cells, lymphocytes have 91-462 units/10(7) cells, and platelets have 1.1-7.1 units/10(7) platelets. These cell differences are discussed with respect to the hypothesis that NTPH prevents incorporation of ITP or dITP into nucleic acids.
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38
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Whelan JM, Bagnara AS. Factors affecting the rate of purine ribonucleotide dephosphorylation in human erythrocytes. BIOCHIMICA ET BIOPHYSICA ACTA 1979; 563:466-78. [PMID: 465500 DOI: 10.1016/0005-2787(79)90065-0] [Citation(s) in RCA: 27] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Purine ribonucleotide dephosphorylation was measured in intact human erythrocytes in vitro to evaluate those factors which might regulate this process in vivo. It was found that purine nucleotides which exist predominantly in the triphosphate form (e.g. ATP and GTP) are protected from dephosphorylation while those nucleotides normally present as the monophosphate (e.g. IMP) are susceptible to dephosphorylation. This point was emphasised by studying an individual whose erythrocytes accumulated ITP rather than IMP; erythrocytes from this individual has a more stable pool of inosine phosphates than did erythrocytes from normal individuals. The concentration of intracellular phosphate was also shown to affect the rate of dephosphorylation. The dephosphorylation of IMP was inhibited at intracellular phosphate concentrations above approx. 3 mM. AMP dephosphorylation (in cells whose AMP concentration was increased by incubating them in the presence of 2-deoxyglucose) was inhibited by phosphate more strongly than was found for IMP. In contrast, the dephosphorylation of GMP did not appear to be affected by phosphate concentration. High oxygen tension was a powerful stimulator of IMP dephosphorylation while low oxygen tension protected IMP from dephosphorylation. This finding shows that human erythrocytes are similar to those of other mammals in this regard and points to a possible physiological determinant of purine turnover in these cells.
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39
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Vanderheiden BS. Inosine di- and triphosphate synthesis in erythrocytes and cell extracts. J Cell Physiol 1979; 99:287-301. [PMID: 457791 DOI: 10.1002/jcp.1040990303] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The ability to synthesize inosinetriphosphate was demonstrated in blood cells as well as in a variety of tissue extracts in spite of the presence of ITP pyrophohydrolase. At the expense of having sub-optimal conditions, an assay system was selected that completely repressed the hydrolyzing enzyme, thus permitting the accumulation of ITP. In an attempt to define the biosynthetic pathway of ITP, and since guanylate kinase has been implicated in the formation of ITP, the rate of synthesis of ITP and GTP in cell extracts was compared. The comparison of the specific activities of the [14C]-labeled hypoxanthine and guanine moieties of the inosine and guanosine phosphates formed during incubation with [8-14C]-inosine and [8-14C]-guanosine respectively, revealed striking differences in the relative rates of isotope incorporation. Tentative mechanisms are proposed to explain these differences. The data obtained thus far does not discard the possibility that ITP may be formed by stepwise phosphorylation and (or) by direct pyrophosphorylation of IMP.
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40
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Zimmerman TP, Deeprose RD. Metabolism of 5-amino-1-beta-D-ribofuranosylimidazole-4-carboxamide and related five-membered heterocycles to 5'-triphosphates in human blood and L5178Y cells. Biochem Pharmacol 1978; 27:709-16. [PMID: 207278 DOI: 10.1016/0006-2952(78)90508-7] [Citation(s) in RCA: 78] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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41
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Henderson JF. Inosine triphosphate metabolism in human erythrocytes. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 1977; 76A:115-20. [PMID: 16444 DOI: 10.1007/978-1-4613-4223-6_15] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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42
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Inhibition of phosphoribosylpyrophosphate synthesis by purine nucleosides in human erythrocytes. J Biol Chem 1976. [DOI: 10.1016/s0021-9258(17)33028-4] [Citation(s) in RCA: 40] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
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