REVIEW

Pharmacogenetic significance of inosine triphosphatase

Jörgen Bierau†, Inosine triphosphatase (ITPase) is the enzyme that catalyzes the conversion of inosine Martijn Lindhout & triphosphate (ITP) and deoxy-inosine triphosphate (dITP) to inosine monophosphate and Jaap A Bakker deoxy-inosine monophosphate, respectively, thereby maintaining low intracellular †Author for correspondence Maastricht University concentrations of ITP and dITP. Individuals deficient in ITPase activity were first recognized Hospital, Laboratory of over 30 years ago. For decades, no clinical significance could be attributed to this inborn Biochemical Genetics, error of metabolism whatsoever. In recent years, evidence has started to accumulate that Department of Clinical Genetics, PO BOX 6202 AZ polymorphisms in the gene encoding ITPase are associated with potentially severe adverse Maastricht, The Netherlands drug reactions towards the thiopurine drugs azathioprine and 6-mercaptopurine. The Tel.: +31 433 877 835; pharmacogenetic significance is presently being debated in the literature. However, the Fax: +31 433 877 901; present state of knowledge is still insufficient to definitively determine the pharmacogenetic E-mail: jorgen.bierau@ gen.unimaas.nl significance of ITPase. This article aims to review the current knowledge on the role of ITPase in thiopurine metabolism. Thiopurines (such as azathioprine, 6-mercapto- and RNA. Purines are involved in numerous cel- purine and 6-thioguanine) are cytotoxic drugs lular processes, for example transport and trans- used for the treatment of patients suffering from fer of energy, signal transduction and the a diversity of immunological and lympho- biosynthesis of cofactors. Purine nucleotides are proliferative diseases. The most frequently used synthesized via two routes (Figure 1). In proliferat- thiopurine-drugs are azathioprine and 6-mercap- ing cells, an energetically costly de novo synthesis topurine (6-MP). Thiopurines are anti-metabo- route is used to synthesize the central purine lites, that is, they inhibit physiological processes nucleotide inosine monophosphate (IMP) from by replacing their natural counterparts (guanos- phosphoribosylpyrophosphate. In resting cells, ine nucleotides). Thiopurines must be activated or cells that do not express the complete de novo by the purine salvage pathway to exert their cyto- pathway, IMP is synthesized by reutilization of toxic effects [1]. The 6-thioguanosine nucleotides the purine base hypoxanthine via hypoxanthine inhibit RNA and DNA synthesis, thereby induc- guanine phosphoribosyl transferase (HGPRT; ing cell death. At least part of the immuno- EC 2.4.2.8). From IMP, the nucleotides inosine suppressive properties of azathioprine is due to diphosphate (IDP) and inosine triphosphate binding of 6-thio-GTP to Rac1 instead of GTP, (ITP) are synthesized by nucleoside monophos- stimulating the mitochondrial apoptotic path- phate kinase and nucleoside diphosphate kinase, way in human CD4+ T lymphocytes [2,3]. respectively. Inosine nucleotides other than IMP A well-known cause of potentially severe thio- may be regarded as by-products of purine purine-induced adverse drug reaction (ADR) is metabolism. No physiological function has yet decreased or absent activity of the enzyme thio- been attributed to these metabolites. purine S-methyltransferase (TPMT). For a long In normal cells, no ITP or deoxy-inosine tri- time, TPMT deficiency was thought to be the phosphate (dITP) is found due to efficient pyro- only enzyme defect causing ADRs in thiopurine phosphohydrolysis of these nucleotides to their Keywords: allelic variant, adverse drug reactions, therapy. However, in recent years evidence has respective monophosphates by ITPase (EC azathioprine, inborn error of been published that polymorphisms in the gene 3.6.1.19). Apparently, IMP, IDP and ITP exist metabolism, inosine encoding inosine triphosphatase (ITPase) may be in a futile cycle that is sustained by the nucle- triphosphatase, inosine triphosphate associated with the occurrence of ADRs in otide kinases and ITPase. This cycle is crucial for pyrophosphohydrolase, patients treated with azathioprine [4,5]. This review maintaining low intracellular levels of ITP and mercaptopurine, will discuss the place of ITPase in thiopurine dITP. Accumulation of ITP may be considered pharmacogenetics, thiopurine, thiopurine methyltransferase metabolism and its pharmacogenetic significance. to be undesirable, as it may interfere in several cellular processes, such as GTP-requiring reac- ITPase in purine metabolism tions [6]. ITP may also interfere in ATP-requiring part of Purines are essential compounds. Together with reactions, for example ITP has been shown to be the pyrimidines, they are the precursors of DNA a substrate for F0-F1-ATPase in vitro [7]. Further-

10.2217/14622416.8.9.xxx © 2007 Future Medicine Ltd ISSN 1462-2416 Pharmacogenomics (2007) 8(9), xxx–xxx 1 REVIEW – Bierau, Lindhout & Bakker

Figure 1. Overview of purine metabolism.

ATP dITP ITP GTP

ADP ITPA dIDP IDP ITPA Purine de novo GDP synthesis

dIMP

AMP IMP GMP AMPDA GMPR

IMPDH GMPS ADSL ADSS S-AMP Ino XMP

PNP HGPRT Hypo XO

Xan XO

Urate

ADSL: Adenylosuccinate lyase; ADSS: Adenylosuccinate synthetase; AMPDA: AMP deaminase; GMPR: Guanosine monophosphate reductase; GMPS: Guanosine monophosphate synthetase; Hypo: Hypoxanthine; HGPRT: Hypoxanthine guanine phosphoribosyltransferase; Ino: Inosine; IMPDH: Inosine monophosphate dehydrogenase; ITPA: Inosine triphosphatase; PNP: Purine nucleoside phosphorylase; S-AMP: Succinyl-adenosine-5´-monophosphate; Xan: Xanthine; XMP: Xanthosine-5´-monophosphate; XO: Xanthine oxidase

more, ITPase deficiency may cause an accumula- exonic splicing-silencing element in exon 2, tion of dITP. dITP is incorporated into DNA by resulting in the activation of two nearby DNA polymerases both in cell lysates and intact upstream 5´ splice sites and mis-splicing of the cell systems [8], thereby rendering DNA-synthe- exons 2 and 3 cassette causing structural changes sis error prone. In fact, dITP is used in to the enzyme, contributing to ITPase site-directed mutagenesis [9]. deficiency [15]. Stenmark and colleagues pre- dicted that the P32T amino acid substitution Inborn error of metabolism resulting from the ITPA c.94C>A mutation Deficiency of ITPase (OMIM 147520) is an causes a structural rearrangement of α-helix 2, inborn error of metabolism and was first pub- possibly leading to a disturbed nucleotide lished over 30 years ago by Vanderheiden [10]. exchange or a partial miss folding of α-helix 2, Enzyme activity assays and catalytic properties possibly rendering the dimer inactive [14]. were published soon thereafter [10–12]. However, A clinical phenotype was expected to be asso- it was not until 2002 that the genetic basis of ciated with ITPase deficiency, and studies were ITPase deficiency was elucidated [6,13]. The crys- performed in patients suffering from mental tal structure of ITPase was published [14] and retardation [17] and schizophrenia [18]. No corre- possible molecular mechanisms of ITPase defi- lation between ITPase deficiency and a clearly ciency were proposed [15,16]. There are two likely defined clinical presentation could be made. The mechanisms of ITPase deficiency resulting from occurrence of ITPase deficiency in a kindred the ITPA c.94C>A mutation. Arenas and col- with adenosine deaminase deficiency proved to leagues proposed that ITPA c.94C>A destroys an be coincidental [19]. The fact that no clear clini-

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cal presentation is associated with ITPase defi- azathioprine therapy (OR: 11.3; 95% CI: ciency is in contrast with other inborn errors of 2.5–50.0; p = 0.001) [26]. Withdrawal at any metabolism directly or indirectly affecting nucle- moment due to azathioprine attributable side otide metabolism. However, in our opinion, it is, effects (myelosuppression, hepatotoxicity, pan- not yet not excluded that ITPase deficiency may creatitis and flu-like symptoms) was also associ- be a modulating factor that may aggravate the ated with the ITPA c.94C>A carrier status (OR: clinical presentation of other defects in nucle- 7.8; 95% CI: 2.1–29.1; p = 0.002) [26]. In otide or nucleic acid metabolism. another study, the ITPA c.94C>A genotype appeared to predict azathioprine-induced myelo- ITPA genetics suppression (OR: 3.504; 95% CI: The gene encoding ITPase, ITPA, is located on 1.119–10.971; p = 0.046), but not the occur- chromosome 20p13 [20]. The gene spans 8 exons rence of hepatotoxicity [27]. Gearry and van and has a length of approximately 13 kb [6]. Dieren found no correlation between ITPA sta- Thus far, five allelic variants have been described tus and any ADR (OR: 1.015; 95% CI: that are associated with decreased enzyme activ- 0.360–2.867; p = 0.593) in their respective stud- ity (Table 1). The possible pharmacogenetic con- ies [28,29]. However, no conclusion could be sequences of these polymorphisms are discussed drawn for ITPA c.94C>A homozygous individu- below. To date, ITPA c.94C>A is the sole allelic als [29]. Allorge found no correlation between variant that may be associated with azathio- ITPA genotype and azathioprine/6-mercaptopu- prine/6-mercaptopurine-induced ADRs. The rine induced myelosuppression, but omitted distribution of ITPA c.94C>A has been studied other ADRs [30]. De Ridder and colleagues by several groups and was reviewed by Marsh found no correlation between ITPA c.94C>A and colleagues [21]. ITPA c.94C>A is found in all and ADRs (leucopenia, pancreatitis and ‘general populations. However, the allele frequency of malaise’) in pediatric patients. Two patients ITPA c.94C>A varies dramatically between pop- homozygous for ITPA c.94C>A tolerated azathi- ulations, being lowest in Central- and oprine well [31]. The study of Breen in which no South-American populations (1–2%) and high- correlation was found between aza- est in Asian populations (11–19%). In Cauca- thioprine-induced ADR and ITPA genotype in sian, African-American and African populations patients who received a liver transplant [32] may the allele frequency of is 5–7% [21]. be due to the fact that the patients’ own geno- The allele frequency of ITPA g.IVS2+21 A>C is type and the genotype of the liver received do approximately 13% in Caucasian populations [6,22] not necessarily correspond. An individual and was not found in a Japanese population [23]. homozygous for ITPA c.94C>A receiving a liver with fully functional ITPase may very well be ITPA allelic variants & rescued from azathioprine-induced toxicity by thiopurine-induced ADR the donor liver. Thiopurine metabolism is an intricate network Thus far, only ITPA c.94C>A was found to be of enzymatic and nonenzymatic reactions and associated with thiopurine associated ADRs by has been reviewed previously [1,24,25]. The signif- some authors. Whether or not an allelic variant has icance of ITPase in thiopurine-based therapeutic pharmacogenetic consequences may be dependent regimens is at present a focal point of discussion. on the residual enzyme activity. ITPA c.94C>A het- In 2004, Marinaki and colleagues published the erozygotes have approximately 25% residual activ- first reports that polymorphisms in ITPA are ity whereas homozygotes have zero activity [4,26]. associated with azathioprine induced ADR [4,5]. However, both heterozygotes and homozygotes for In a retrospective study of patients suffering ITPA g.IVS2+21A>C, and ITPA g.IVS2+68T>C from inflammatory bowel disease (IBD), the have significant residual activity (≥60%) [4,33]. occurrence of side-effects such as rash (odds ratio These genotypes have not been found to be associ- [OR]: 10.3; 95% confidence interval [CI]: ated with ADRs. ITPA g.IVS2+68T>G [23] and 4.7–62.9; p = 0.0213), flu-like symptoms (OR: ITPA 359_366dupTCAGCACC [22] are more 4.7; 95% CI: 1.2–18.1; p = 0.0308) and pancre- likely to have pharmacogenetic significance, as het- atitis (OR: 6.2; 95% CI: 1.1–32.6; p = 0.0485) erozygotes have similar residual activity to ITPA was significantly correlated with ITPA c.94C>A c.94C>A heterozygotes [4,26], and zero residual [4]. In a prospective study by von Ahsen and activity can be expected in homozygotes. colleagues, patients carrying ITPA c.94C>A were Since ITPase is not involved in the catabolism significantly more likely to withdraw early from of 6-thioguanine, the use of 6-thioguanine futurefuture sciencescience groupgroup www.futuremedicine.com 3 REVIEW – Bierau, Lindhout & Bakker

Table 1. Allelic variants of ITPA associated with decreased enzyme activity. Genotype Residual enzyme activity Association with ADR reported Ref.

ITPA c.94C>A Heterozygote: 22.5% Yes [4,6] Homozygote: <1% ITPA g.IVS2+21 A>C Heterozygote: 60% No [4,6] ITPA g.IVS2+68T>G Heterozygote: 30% Not yet reported [23] ITPA g.IVS2+68T>C Heterozygote: 60% No [33] ITPA 359_366dupTCAGCACC Heterozygote: 30% Not yet reported [22] ADR: adverse drug reaction; ITPA: Inosine triphosphatase

instead of azathioprine for individuals with thio-ITP may occur in cells lacking ITPase activ- ITPase deficiency would from a biochemical ity when challenged with 6-MP. This is point of view be an alternative. However, in real- supported by the finding that 6-methylthio-ITP, ity 6-thioguanine gives rise to many serious side- but not 6-thio-ITP is detected in (white blood) effects and is therefore less attractive. When cells obtained from bone marrow aspirates and 6-thioguanine is used it is of the utmost impor- peripheral blood samples [36,37]. This indicates tance to be aware of the TPMT status of the that the 6-thioinosine nucleotides are substrates patient. One should bear in mind that thiopu- for TPMT. Further detailed in vitro studies of rine metabolism is complex and is at present far the metabolism of (methyl)thiopurine nucle- from being fully elucidated. otides are needed to further elucidate role of ITPase. Preferably, the thiopurine nucleotide dis- ITPase substrate specificity & its tribution should also be studied in peripheral implications blood cells obtained from individuals treated The fact that ITPase catalyzes the pyrophospho- with 6-MP or azathioprine. Although erythro- hydrolysis of ITP, dITP and xanthosine-5´-tri- cytes are not capable of purine de novo synthesis, phosphate is well established [11,12,34]. However, their nucleotide content is thought to reflect the while the assumption that ITPase catalyzes the nucleotide status of the liver and other tissues [1]. pyrophosphohydrolysis of the 6-MP metabolite Whilst high performance liquid chromatogra- 6-thio-ITP to 6-thio-IMP is generally made, it phy (HPLC) analysis of hydrolyzed thiopurine has never been demonstrated that 6-thio-ITP is nucleotides contained within erythrocytes is use- actually a substrate for ITPase. We have deter- ful for therapeutic drug monitoring [38,39], it pro- mined the substrate specificity of human vides no information on the fractions of the erythrocyte ITPase towards 6-thio-ITP and nucleotides present. Only by meticulous study of 6-methylthio-ITP, and compared it to the sub- natural purine nucleotides and thiopurine nucle- strate specificity of ITP. As shown in Table 2, ITP otides in human cells can the pharmacogenetic is the preferred substrate for human erythrocyte significance of ITPase be elucidated. ITPase. 6-thio-ITP is also efficiently converted to 6-thio-IMP, whereas 6-methylthio-ITP was a Expert commentary poor substrate. Michaelis-Menten kinetics were The results of clinical studies investigating the observed for all substrates. Together with the pharmacogenetic significance of ITPase in thio- previous observation that RNA polymerase is purine-based therapy are contradicting. Correct inhibited by 6-thio-ITP [35] our findings are in assessment of the pharmacogenetic significance support of the possible pharmacogenetic signifi- of ITPase is hampered by the small number of cance of ITPase. We propose the metabolism of patients in each study and the different 6-MP and azathioprine as depicted in Figure 2. A approaches used. More extensive and preferably secondary increase in accumulation of 6-methyl-

Table 2. Substrate specificity of human erythrocyte ITPase.

Substrate KM (µM) Vmax (µmol/mg protein/h) Vmax/KM Inosine-5´-triphosphate 107 1068 9.9 6-thio-inosine-5´-triphosphate 283 512 1.8 6-methylthio-inosine-5´-triphosphate 924 335 0.4

4 Pharmacogenomics (2007) 8(9) futurefuture sciencescience groupgroup Pharmacogenetic significance of inosine triphosphatase – REVIEW

Figure 2. Putative involvement of ITPAse in the metabolism of azathioprine.

6-Me-TITP

No ITPA catalyzed 6-Me-TIDP pyrophosphohydrolysis

6-Me-TIMP 6-MMP

TPMT TPMT IMPDH GMPS AZA 6-MP 6-TIMP 6-TXMP 6-TGMP HGPRT

ITPA NMPK

? 6-Me-TITP 6-TITP 6-TIDP TPMT NDPK

6-MP: 6-mercaptopurine; 6-TIMP, 6-TIDP; 6-TITP: 6-thio-inosine-5´-mono-, di and triphosphate; respectively, 6-Me-TIMP; 6-Me-TIDP, 6-Me-TITP: 6-methylthio-inosine-5´-mono-; di and triphosphate, respectively; 6- TXMP: 6-thio-xanthosine-5´-monophosphate, 6-TGMP: 6-thio-guanosine-5´-monophosphate; Aza: Azathioprine; GMPS: Guanosine monophosphate synthetase; HGPRT: hypoxanthine guanine phosphoribosyltransferase, IMPDH: inosine monophosphate dehydrogenase; ITPA: Inosine triphosphatase, NDPK: Nucleoside diphosphate kinase; NMPK: Nucleoside monophosphate kinase; TPMT: Thiopurine S- methyl transferase

prospective studies are needed to definitively activity, we recommend monitoring of cellular determine the pharmacogenetic significance of blood count and liver function. Subsequently, ITPase. At present it is to early to dismiss or fully molecular diagnostics are used to confirm the accept the pharmacogenetic value of ITPase. results of the enzyme activity assay. If ITPA Although screening for the most frequent g.IVS2+21A>C is found, it is reported that this allelic variant for both ITPA and TPMT genotype has not yet been associated with azathi- accounts for the vast majority of reduced activi- oprine or 6-MP-induced ADR. In case of ties, the chance that patients with rare or novel reduced TPMT activity in combination with mutations will be missed cannot be ignored. The reduced or absent ITPase activity, therapeutic enzymatic activities of TPMT and ITPase are drug monitoring is also recommended. In the easily measured and a clear correlation between event that TPMT deficiency is established, the genotype and enzyme activity exists [4,6,33]. In use of thiopurines is strongly discouraged, our laboratory, we therefore offer both ITPase regardless of the ITPase status. and TPMT enzyme activity assays as one diag- nostic package. Based on the initial reports of Future perspective Marinaki and colleagues [4,5] and our own find- In the years to come, the pharmacogenetic sig- ings we decided to implement the ITPase activity nificance of ITPase in thiopurine-based therapy assay. For the past 2 years, ITPase activity has will become more clear. Based on substantial been offered as an additional service to the physi- clinical studies that remain to be published, cian and the patient. The physician is informed ITPase as a risk-factor for thiopurine induced that the patient may have a decreased tolerance ADRs will either be generally accepted or abol- for azathioprine and 6-MP when the ITPase ished. Furthermore, there is a dire need for more activity measured is in the carrier range or defi- biochemical data. cient. Awareness of the side effects reported in The interactions between ITPase and the other the literature is raised. In all cases of reduced key enzymes in thiopurine metabolism need to be futurefuture sciencescience groupgroup www.futuremedicine.com 5 REVIEW – Bierau, Lindhout & Bakker

extensively studied. It is not unthinkable that cer- Decreased activity of both IPMDH and ITPase is tain ‘favorable’ and ‘unfavorable’ combinations of expected to lead to a massive accumulation of activities and/or polymorphisms in the other pro- 6-thio-ITP, of which the consequences are still teins involved in purine, DNA and RNA metabo- unknown. lism will become clear. The interactions between Pharmacogenetic significance of ITPase may ITPase and TPMT and ITPase and IMPDH1 in theory be expected for all therapies based on will be worthwhile to study as they will determine inosine analogs. One of these analogs is didanos- the balance between (methyl)thiopurine metabo- ine (2´,3´-dideoxyinosine), an antiretroviral lites and the cytotoxic 6-thio-guanine metabolites. drug. Preliminary results from our laboratory The interaction between ITPase and IMPDH1 is show that didanosine is a substrate for human particularly interesting, as one patient resistant to erythrocyte ITPase. It may be that the true azathioprine therapy has been described with a pharmacogenetic significance of ITPase is to be mutation in the IMPDH P3 promoter [40]. found in antiretroviral therapy.

Executive summary

• The pharmacogenetic significance of inosine triphosphatase is a highly debated subject and data published are contradictory.

• At present the pharmacogenetic significance of ITPase can neither be established nor dismissed.

• ITPA is a polymorphic gene.

•The ITPA 94>A genotype may be associated with azathioprine induced adverse drug reaction.

• Azathioprine-induced ADRs-associated with ITPase deficiency may be flu-like syndrome, rash, pancreatitis, myelotoxicity and hepatotoxicity.

• In Western and Asian populations the ITPA 94>A allele frequency is 5–19%.

• ITPase activity is easily measured in erythrocytes.

•Carriers of ITPA allelic variants are easily detected by measurement of ITPase activity.

Appendix Materials & methods

The substrate specificity of human erythrocyte 5´-triphosphate was measured using of 0.6 mg ITPase towards ITP (Sigma-Aldrich, Zwijn- of protein. The reaction was started by addition drecht, The Netherlands), 6-thio-inosine-5´- of protein to the assay mixture. The reaction triphosphate and 6-methylthio-inosine-5´-tri- was incubated in shaking water bath at 37ºC phosphate (Jena Bioscience, Jena, Germany) for 30 min. The reaction was terminated by was determined in a pooled erythrocyte lysate acid precipitation with perchloric acid and obtained from multiple anonymous donors. incubated for 10 min on ice. The sample was The assay was essentially performed as previ- centrifuged and the supernatant was transferred ously described [41]. Briefly, pyrophosphohy- to a clean vial and neutralized using K2CO3. drolase activity was measured in a final volume The precipitate was removed by centrifugation of 200 µl containing 100 mM Tris-HCl pH and the supernatant was diluted twofold in 8.5, 50 mM MgCl2, 0.5 mM dithiothreitol and HPLC-elution buffer and centrifuged over a 31–2500 µM ITP, 6-thio-inosine-5´-triphos- 0.2 µm nylon filter. HPLC analysis was per- phate or 6-methylthio-inosine-5´-triphosphate, formed using an ion-pair chromatography. The respectively. The pyrophosphohydrolysis of products were detected using a DAD-detector ITP and 6-thio-inosine-5´-triphosphate was and calibrated using external standards. Appar- measured using of 0.2 mg of protein, the pyro- ent KM and Vmax values were deduced from phosphohydrolysis of 6-methylthio-inosine- Lineweaver-Burk plots.

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