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Mutation Research 753 (2013) 131–146

Contents lists available at ScienceDirect

Mutation Research/Reviews in Mutation Research

jo urnal homepage: www.elsevier.com/locate/reviewsmr

Co mmunity address: www.elsevier.com/locate/mutres

Review

ITPA (inosine triphosphate pyrophosphatase): From surveillance of

nucleotide pools to human disease and pharmacogenetics

a a,b,d,e a,b,c,

Peter D. Simone , Youri I. Pavlov , 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

Department of Pathology and Microbiology, University of Nebraska Medical Center, USA

Department of Genetics, St-Petersburg University, St-Petersburg, 199034, Russia

A R T I C L E I N F O A B S T R A C T

Article history: Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a

Received 6 May 2013

plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily

Received in revised form 31 July 2013

conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs

Accepted 2 August 2013

inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form

Available online 19 August 2013

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

Keywords:

leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA

Nucleotide pool

deﬁciency in tissues and plays a role in patients’ response to drugs. Structural analysis of this mutant

ITPA gene polymorphism

Pharmacogenetics protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The

Base analogs Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active

Mercaptopurines enzyme monomer targets both homo- and heterodimers to degradation.

Dominant negative

Contents

1. Introduction ...... 132

2. ITPA history, function, and structure ...... 132

2.1. A brief history of ITPA ...... 132

2.2. ITPA function and structure ...... 132

3. Origins of non-canonical nucleotides ...... 135

4. Problems arising from non-canonical nucleotides ...... 136

5. Effects of ITPA deﬁciency in model organisms ...... 138

5.1. E. coli...... 138

5.2. Yeast ...... 138

5.3. Mice...... 138

6. ITPA deﬁciency in humans ...... 139

6.1. Human ITPA polymorphism and the Pro32Thr variant ...... 139

6.2. Potential implications of ITPA deﬁciency in human disease...... 140

6.3. ITPA pharmacogenetics...... 142

7. Concluding remarks...... 144

Acknowledgements ...... 144

References ...... 144

Abbreviations: ITPA, inosine triphosphate pyrophosphatase; ITP, inosine triphosphate; XTP, xanthosine triphosphate; HAM1, Saccharomyces cerevisiae; ITPA, homolog RdgB E. coli

ITPA homolog (Rec-dependent growth B); HAP, base analog 6-hydroxylaminopurine; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; TPMT, thiopurine S-

methyltransferase; SSB, single-strand break; DSB, double-strand break; MEF, mouse embryonic ﬁbroblasts; NUDT16, nudix (nucleoside diphosphate linked moiety X)-type

motif 16.

* Corresponding author at: The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 987696 Nebraska Medical Center,

Omaha, NE 68198-7696, USA. Tel.: +1 402 559 8578; fax: +1 402 559 3739.

E-mail address: [email protected] (Gloria E.O. Borgstahl).

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132 P.D. Simone et al. / Mutation Research 753 (2013) 131–146

1. Introduction ITP, dITP and XTP. With the sequence of the gene in hand, the level

of ITPA expression in various tissues was measured with northern

Maintenance of cellular nucleotide pools is critical for preserving blots. Expression varied greatly among the 24 tissues tested. The

the integrity of the genome and for allowing normal cellular highest levels were found in the heart, liver, pancreas, thyroid,

processes to occur. Inosine triphosphate pyrophosphatase (ITPA) is testis/ovary, and adrenal glands. The puriﬁcation of recombinant

one of several enzymes whose job is to cleanse the nucleotide pool. ITPA made the enzyme easily available and set the stage for future

ITPA acts on the non-canonical purines inosine and xanthosine developments, including the identiﬁcation of additional sub-

triphosphate (ITP/XTP) and their deoxy forms (dITP/dXTP) [1]. ITPA strates, such as the triphosphates of mutagenic base analogs 6-

has homologs in all domains of life. Further proof of its importance hydroxylaminopurine (HAP) and 2-amino-6-hydroxylaminopur-

was demonstrated by the negative effect of mutations in the ITPA ine (AHA) [15].

orthologs in model organisms [2,3]. A particularly dramatic example The last major advancement was the crystallization of ITPA and

was seen in Itpa knockout mice [4]. Loss of ITPA activity in humans the subsequent visualization of its structure in detail. Open,

has been implicated in disease and in the degree of adverse reactions nucleotide-free ITPA was solved independently by Porta et al. in

of patients to several drugs [5,6]. A polymorphism responsible for 2006 [16] and Stenmark et al. in 2007 [17]. Stenmark and

the most dramatic reduction in ITPA activity leads to a proline to coworkers also solved a crystal structure of ITPA with ITP bound.

threonine substitution at amino acid number 32 [7,8]. In vitro studies Together, these structures show the conformational changes upon

of human cells containing this variant have conﬁrmed that ITPA is ligand binding, help explain the speciﬁcity of ITPA for the non-

necessary for protecting the genome from damage [9]. The crystal canonical purines ITP and XTP and explain why ITPA does not seem

structure of Pro32Thr ITPA helps explain its dominant negative to discriminate between NTPs and dNTPs. These aspects will be

effect on enzymatic activity in cells [10]. described next.

2.2. ITPA function and structure

2. ITPA history, function, and structure

ITPA catalyzes the following reactions:

2.1. A brief history of ITPA

Mg2þ

In 1964 Liakopoulou and Alivisatos discovered the presence of an ðdÞITP þ H2O ! ðdÞIMP þ PPi

Mg2þ

ITPase in human erythrocytes [11]. This ITPase activity was

ðdÞXTP þ H2O ! ðdÞXMP þ PPi

separable from ATPase activity, dependent on the presence of

Mg ions and competitively inhibited by adenine derivatives. In The optimal parameters for maximal activity have varied

1970, Vanderheiden demonstrated by using partially puriﬁed somewhat among different studies, but in general ITPA has an

preparations of the ITPase from human erythrocytes that the alkaline pH optimum (8.5 or greater) and a requirement for Mg

enzyme released PPi and therefore was an ITP pyrophosphatase [12]. ions, with around 50 mM providing the greatest activity [1,13,14].

Then, in 1979, this ITP pyrophosphatase was puriﬁed 2,300-fold The kinetic parameters, viz. the Michaelis constant (Km), the

from human erythrocytes and was then characterized in terms of maximum rate (Vmax), and turnover number (kcat), have also varied

kinetics, optimal reaction conditions, and substrate speciﬁcity. ITPA to a degree among reports (Table 1).

was found to be highly speciﬁc for ITP, with only minimal activity Differences in the kinetic parameters listed in Table 1 are likely

against ATP, GTP, CTP, and UTP [13]. In the same year, Holmes and due to the different sources, purity levels and the assay used. Early

coworkers studied the tissue distribution and subcellular localiza- studies were done using ITPA puriﬁed from erythrocytes at various

tion of ITPA. They found that there was large variation between levels of purity. Later studies used highly puriﬁed recombinant

individuals in the level of ITPA activity. In general erythrocytes had human ITPA [1,15,18,19]. It is possible that ITPA is post-

low levels of activity and other cell types, such as bone marrow translationally modiﬁed and that the lack of these modiﬁcations

ﬁbroblasts, had substantially greater activity. They also localized in a recombinant system may alter the kinetics [1]. The assay

ITPA activity to the soluble cytoplasmic fraction of cells. Lastly they method likely has an effect on the values obtained. For example,

conﬁrmed Vanderheiden’s results that ITPA was highly speciﬁc for Vanderheiden [13] used radiolabeled ITP as the substrate and

ITP and dITP, and also demonstrated that XTP was a substrate [14]. directly measured IMP with high-voltage paper electrophoresis

These would be the last major developments in human ITPA and liquid scintillation counting [20]. The studies of Holmes [14],

characterization until the turn of the century. Lin [1,15] and Stepchenkova [18], used a coupled assay where the

In 2001, the human ITPA gene was cloned and functionally PPi produced by ITPA is further broken down by inorganic

expressed in E. coli [1]. The human ITPA gene was localized to pyrophosphatase to Pi, which is then measured colorimetrically.

chromosome 20p. Puriﬁed recombinant ITPA speciﬁcally cleaved This assay assumes that the inorganic pyrophosphatase reaction is

Table 1

Summary of studies on ITPA catalytic properties.

Reference Liakopoulou [11] Vanderheiden, 1970 [12] Vanderheiden, 1979 [13] Holmes [14] Lin [1] Stepchenkova [18] Herting [15,19]

Protein source Hemolysate Partially purified Purified from Hemolysate Purified from Purified from Purified from

from hemolysate hemolysate E. coli E. coli E. coli

4 4 4 5 4 4 5

Km 1 10 M 6 10 M 1.3 10 M 7 10 M 5.1 10 M 4.0 10 M 3.25 10 M

Vmax N/A N/A 1.2 10 M/min N/A 1520 mmol/ N/A N/A

(min mg)

1 1 1

kcat N/A N/A N/A N/A 580 s 91 s 79.6 s

pH optimum 7.5 8.5 (9.5 with DTT) 8.6 in Tris–HCl 9.6 N/A 10.0 N/A N/A

in glycine

Mg optimum 50 mM 50 mM 100 mM 60 mM 30–100 mM N/A N/A

Substrate inhibition No Yes Slight No Yes Yes Yes

Reducing agent N/A Yes No Yes Yes N/A N/A

activation

The substrate was ITP in all cases except for Herting, where dITP was used.

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P.D. Simone et al. / Mutation Research 753 (2013) 131–146 133

not rate limiting. Further, ITP is known to inhibit human inorganic of the DNA mismatch repair enzyme complex Msh2-Msh6 found

pyrophosphatase [21] and may therefore account for the substrate that about 100 Cd ions were bound per protein complex, likely

inhibition observed in some studies (see Table 1). Given the binding to Cys and Met side chains, amino and carbonyl groups,

inhibitory action of IDP on ITPA (see below), the presence of IDP in and so on, leading to non-speciﬁc inhibition [23]. This is likely the

2+ 2+

the ITP used as substrate could also account for substrate case with ITPA. Cu is a borderline acid and Ca is a hard acid [24].

inhibition, as suggested by some authors [1,14]. Lastly, the various This could explain the similar, but slightly reduced inhibition from

2+ 2+ 2+

buffer systems and different reaction conditions used likely Cu compared to Cd , and Ca is thus likely to inhibit by a

account for some variability, especially given the different pH different mechanism, such as competing for the Mg binding site.

optimum observed by Vanderheiden for glycine versus Tris-HCl The ITPA monomer is 21.5 kDa and is composed of 194 amino

buffers. acids. A homodimeric quaternary structure was ﬁrst observed by

Various inhibitors of the ITPA reaction have been identiﬁed. gel ﬁltration, where the calculated mass was twice that expected

When ITPase activity was originally discovered, adenine, adeno- for a monomer [1]. The dimeric nature of ITPA was observed in all

sine, and the mono-, di-, and triphosphate forms were found to crystal structures [16,17]. The protein consists of a long central b-

inhibit the activity in a competitive fashion [11]. However, later sheet forming the ﬂoor of the active site, with two mainly a-helical

work by Holmes and colleagues found that adenosine and its lobes ﬂanking the active site (upper and lower lobes, Fig. 1A). The

nucleotides had very little effect on the activity of ITPA [14]. They monomers in the dimer are related by a 2-fold symmetry axis. The

showed, though, that IDP is a strong competitive inhibitor of the dimer interface involves mainly the upper lobe, with a buried

5 2

ITPA reaction, with a dissociation constant of 1.2 10 M. Several surface area of 1080 A˚ and involves 9 hydrogen bonds, 9 bridging

divalent cations were found to also inhibit ITPA [13]. In the waters, an aromatic ring-stacking interaction, and numerous

2+ 2+ 2+

presence of 50 mM Mg , as little as 1 mM Cd and Cu inhibited hydrophobic contacts (Fig. 1B) [17]. This extensive interface holds

the enzyme by 62% and 40%, respectively. Ca also functions as an the dimer together, even in dilute solutions, and the enzyme

inhibitor, but a concentration of 10 mM was required to give functions as a dimer [18].

inhibition. Cd is a soft Lewis acid and therefore interacts well The structure with ITP bound reveals that the substrate binds in

with soft bases, such as sulfur atoms [22]. A study of Cd inhibition the cleft between the two lobes (Fig. 1A). Upon substrate binding,

Fig. 1. The structure of human ITPA. (A) The structure of ITPA (PDB code 2CAR [17]) without substrate. The protein is composed of a central b-sheet (yellow) with the active

site ﬂanked by two predominantly a-helical lobes: an upper lobe in light blue and a lower lobe in red. (B) In the ITPA dimer the monomers are related by a 2-fold symmetry

axis. The majority of the dimerization interactions occur in the upper lobe (light blue). Residues in the interface are colored as follows: glutamic acid = green,

glutamine = purple, lysine = gray, leucine = brown, phenylalanine = red, tryptophan = orange, tyrosine = dark blue. This interface buries 1080 A˚ of surface area and involves

numerous hydrophobic contains and hydrogen bonds. (C) Stereopair of the structure of ITPA with ITP bound (PDB code 2J4E [17]), shown in purple, pink, and green, reveals

several structural changes upon substrate binding. The most prominent is in the lower lobe with the closing of the central a-helix towards ITP, indicated by the arrow. The a-

carbons of the two structures align with an r.m.s.d. of 1.87 A˚ . Ribbon ﬁgures were generated with PyMOL [134]. (For interpretation of the color information in this ﬁgure

legend, the reader is referred to the web version of the article.)

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134 P.D. Simone et al. / Mutation Research 753 (2013) 131–146

Fig. 2. Substrate binding and catalysis. (A) Stereopair of ITPA with ITP bound (PDB code 2J4E [17]) is shown with relevant residues involved in substrate binding and

discrimination shown in stick form. A Mg cation is shown as a green sphere. (B) Potential reaction mechanisms for ITPA are shown schematically. Due to the low resolution

of the structure, a single catalytic mechanism cannot be determined. Asp 72 is in position to coordinate a water molecule for attack on the a phosphate (circled on the top),

while Asn-16 is positioned to coordinate a water for attack on the b phosphate (circled on the bottom). Either would result in the scission of the a-b phosphoanhydride bond.

The positively charged e-amino groups of Lys 19 and Lys 89 are positioned to stabilize the negatively charged intermediate. (For interpretation of the color information in this

ﬁgure legend, the reader is referred to the web version of the article.)

the enzyme shifts to a closed position in which the lower lobe toward the solvent (Fig. 2A). This explains the ability of ITPA to act

moves 258 towards the upper dimerization lobe. Most notable is on both NTPs and dNTPs [17].

the movement of the a-helix formed by residues 16 to 27 toward Lastly, the structure of ITPA with bound ITP allows for a

the substrate (Fig. 1C, arrow). The nucleotide base is clamped by potential reaction mechanism to be proposed (Fig. 2B). Unfortu-

ring-stacking interactions involving Phe149 and Trp151 (Fig. 2A; nately the resolution of 2.8 A˚ prevents exact details, such as the

Table 2). The Mg cation is coordinated by the b and g phosphates

of ITP and the side chain of Glu44. Substrate speciﬁcity is explained Table 2

as follows. Lys172, His177, and Arg178 are within hydrogen Assignment of function of active site residues in human ITPA.

bonding distance of the 6-keto oxygen of ITP and XTP. ATP contains

Role Residues

an amino group at this position which would not allow for

Inosine ring stacking Phe149, Trp151

hydrogen bonding. Although the protein was not crystallized with

Inosine speciﬁcity Asp152, Lys172, His177, Arg178

XTP, a potential hydrogen bond can be identiﬁed between the 2-

Ribose Asn16

keto oxygen and the backbone amide of Asp152. GTP is excluded a-phosphate Lys19, Glu44/Mg , Asp72

from the active site because an amino group in this position would b-phosphate Asn16, Lys19, Glu44/Mg , Lys89

g-phosphate Thr14, Gly 15, Glu44/Mg , Lys56

not be accommodated. Neither of the ribose hydroxyl groups

makes strong contacts with the protein and instead point out Strictly conserved residues are shown in bold.

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P.D. Simone et al. / Mutation Research 753 (2013) 131–146 135

role of water, from being described [17]. The nucleotide is stably group to an amino group. For this to occur, aspartate is connected

bound in the active site in a conformation amenable for catalysis. by its amino group to the C6 of IMP by adenylosuccinate

Two possibilities exist for the location of the hydrolytic water. synthetase in a GTP-requiring reaction. Then adenylosuccinate

Asp72 could coordinate a water molecule for nucleophilic attack lyase cleaves off fumarate, leaving AMP. To form GMP the C2 of IMP

on the a-phosphate, whereas Asn16 is positioned to coordinate a must ﬁrst be oxidized to a keto group by IMP dehydrogenase with

water molecule for attack on the b-phosphate. In either case, the the concomitant reduction of NAD to NADH forming XMP. In the

result is the scission of the a-b phosphoanhydride bond. The second step, the keto group in converted to an amino group by

residues Lys89 and Lys19 are in a position such that they could GMP synthetase using glutamine and ATP [28].

stabilize the negatively charged intermediate of the reaction. IMP, AMP, and GMP are also formed from the purine bases

Clearly a higher resolution structure is needed to resolve the hypoxanthine, adenine, and guanine, respectively, by the purine

speciﬁcs of the reaction. salvage pathway (Fig. 3). These bases are formed during normal

Structures of ITPA analogs from other organisms have been nucleic acid turnover, and the salvage pathway allows them to be

solved, including Thermatoga maritima (PDB code 1VP2), Pyrococ- reused, thus circumventing the need for the energetically costly de

cus horikoshii (PDB codes 1V7R, 2DVP, 2DVO, 2DVN, and 2ZTI [25]), novo pathway. No matter which pathway is used, once AMP and

Methanococcus jannaschii (PDB codes 1B78 and 2MJP [26]), and E. GMP are formed, the two additional phosphate groups are added

coli (PDB codes 1K7K, 2PYU, and 2Q16 [27]). These structures show sequentially by base-speciﬁc nucleoside monophosphate kinases,

a remarkable degree of conservation of tertiary structure. In all forming ADP and GDP, and nucleoside diphosphate kinase,

cases the structure forms a 2-fold symmetric homodimer with a producing the triphosphate forms. Lastly, the nucleoside dipho-

large buried surface area of about 1000 A˚ . These structural sphates (NDPs) can be converted to the corresponding deoxynu-

similarities indicate that ITPA and its homologs are performing an cleoside diphosphates (dNDPs) by ribonucleotide reductase. The

important and evolutionarily conserved function. dNDPs can then be phosphorylated by NDP kinase to dNTPs for use

in DNA synthesis [28]. Although these reactions can potentially

3. Origins of non-canonical nucleotides directly convert IMP and XMP to ITP and XTP, their impact on the

overall level of noncanonical triphosphates in living cells is not

Given that a ubiquitous enzyme exists to remove the non- known.

canonical nucleotides ITP and XTP, the question that arises is how Early studies showed that erythrocytes were capable of

they are formed in the ﬁrst place. IMP is the ﬁrst nucleotide synthesizing ITP from inosine [29,30]. Two possibilities for the

produced in the de novo synthesis pathway by a series of eleven initial formation of IMP were suggested: (1) the direct phosphor-

reactions (Fig. 3). IMP can be used for either the synthesis of AMP or ylation of inosine by an ‘‘inosine kinase’’ and (2) via free

GMP. The formation of AMP from IMP requires changing the 6-keto hypoxanthine generated by the phosphorolytic cleavage of inosine

Fig. 3. Purine metabolism and the formation of ITP and XTP. IMP is the ﬁrst purine nucleotide formed in the de novo synthesis pathway. IMP is converted to either AMP or GMP,

the latter through XMP as an intermediate. IMP and GMP can also be made from the free bases hypoxanthine and guanine by hypoxanthine-guanine

phosphoribosyltransferase (HGPRT) in the salvage pathway. Similarly, AMP can be synthesized from adenine by adenine phosphoribosyltransferase (APRT). AMP and

GMP are sequentially phosphorylated to ATP and GTP by nucleoside monophosphate kinases and nucleoside diphosphate kinase. These enzymes may also work on IMP and

XMP, providing one means of generating ITP and XTP. Highlighted in green are the functional groups that differ between ATP and ITP and between GTP and XTP. ATP and GTP

contain amino groups, while ITP and XTP contain keto groups. The amino groups can undergo deamination to keto groups. Hence, this deamination of ATP and GTP is another

mechanism that can produce ITP and XTP. (For interpretation of the color information in this ﬁgure legend, the reader is referred to the web version of the article.)

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136 P.D. Simone et al. / Mutation Research 753 (2013) 131–146

by purine nucleoside phosphorylase. They also suggested that the adverse effects (Fig. 4). Several assumptions are implicit here. First

following two phosphorylation steps are performed by nucleoside is that non-canonical nucleotides can base pair with the canonical

monophosphate kinase and NDP kinase with ATP serving as the ones. It is known, for example, that inosine can form Watson-Crick

phosphate donor. Next researchers found that ITP could be base pairs with cytosine, although only two hydrogen bonds are

synthesized from hypoxanthine [31–33] favoring the second formed and their interaction is weaker than standard canonical

theory for the generation of IMP. All of these studies focused on base pairs [41]. Another assumption is that RNA and DNA

erythrocytes. polymerases are able to utilize (d)ITP and insert it into the nascent

In 1979, Vanderheiden sought to examine the enzymatic strand. Human RNA polymerase II has been shown to incorporate

mechanism for ITP formation in a variety of cells [34]. Using inosine opposite cytosine with a similar Km and Vmax as for

conditions that inhibit the activity of ITPA, levels of IMP, IDP, and ITP inserting the canonical guanine. However, the incorporation of

were measured over time and compared to the levels of GMP, GDP, inosine inhibited elongation of the chain, allowing for the

and GTP in cells incubated with guanosine. The rates of formation proofreading action of RNA polymerase II [42]. It has also been

and disappearance of IMP, IDP, and ITP differed from those of GMP, found that HeLa nuclei and partially puriﬁed DNA polymerase a

GDP, and GTP, indicating differences in the enzymatic pathways are able to incorporate dITP, although at a substantially reduced

used or in the afﬁnity of the enzyme for the particular nucleotide. rate compared to dGTP. Also of note, in the reaction dITP could

Unfortunately, the data could be interpreted two ways: (1) that ITP is substitute for dGTP, but not dATP [37,43,44]. Major yeast

formed by the sequential phosphorylation of IMP or (2) that ITP is replicative DNA polymerases d and e incorporate dITP opposite

formed by the direct pyrophosphorylation of IMP. Further experi- template C only 2-4 fold less efﬁciently than the correct dGTP, and

ments are therefore needed to unequivocally establish the only polymerase e can incorporate dITP opposite template T (a

enzymatic pathway from inosine or hypoxanthine to ITP. putative mutagenic intermediate) but with a hundred fold less

There has been some evidence along these lines. Agarwal et al. efﬁciency than the correct dATP (C. Grabow and Y. Pavlov,

found that IMP can serve as a substrate for human erythrocyte unpublished data). It is difﬁcult to estimate the rate of incorpo-

guanylate kinase to form IDP [35]. However, the reaction is very ration of dITP in vivo, because it is present at low concentration in

slow, with a Vmax approximately 0.2% that of the reaction for GMP. the pool of precursors and has to compete with normal

The Km was also two orders of magnitude higher for IMP than for nucleotides. Nevertheless, in sensitized genetic backgrounds one

GMP. More recently, human adenylate kinase-6 was shown to inosine per 10,000 bases is detected in yeast DNA [45]. DNA

phosphorylate IMP, but, as with guanylate kinase, to a much lower polymerases can also incorporate dXTP opposite C albeit at very

extent than its preferred substrate (d)AMP [36]. A study with HeLa low efﬁciency [46]. This is consistent with xanthosine also forming

cell nuclear extracts showed that dIDP is rapidly converted to dITP a Watson-Crick base pair with cytosine. The incorporation of (d)ITP

in the presence of ATP, presumably by NDP kinase [37]. The high into RNA and DNA was found in ITPA knockout organisms and will

ATP levels in cells may be driving the following reactions towards be described in detail later.

the formation of ITP: The presence of noncanonical bases in RNA or DNA has

deleterious effects. Their presence in RNA could affect the structure

Guanylateðor adenylateÞkinase

IMP þ ATP!IDP þ ADP of large RNA complexes, such as the ribosome, or could lead to

NDP kinase

mistranslation of proteins by altering the codons of mRNA or

IDP þ ATP!IDP þ ADP:

changing the properties of tRNA [47]. In terms of effects on DNA,

Finally, there is another mechanism for the formation of (d)ITP the most obvious would be the induction of mutations. Incorpo-

and (d)XTP. (d)ATP contains a 6-amino group while (d)ITP has a keto ration of dITP is non-mutagenic per se, because dITP is

group in the same position. Similarly, (d)GTP contains a 2-amino predominantly incorporated opposite cytosine and behaves like

group whereas (d)XTP has a 2-keto group. Deamination of the amino G in subsequent replication cycles [48]. However, when cellular

groups can thus convert (d)ATP to (d)ITP and (d)GTP to (d)XTP. systems attempt repair of incorporated dITP, DNA breaks are

Nitrous acid is known to react with adenine and guanosine, resulting produced as intermediate products that can lead to induction of

in the formation of hypoxanthine and xanthine, respectively [38]. recombination and chromosomal rearrangements in sensitized

Other mechanisms of deamination include hydrolytic deamination genetic backgrounds (see below) [49,50]. Because dXMP in DNA is

and nitrosative deamination by reactive nitrogen species produced difﬁcult to bypass, it can cause DNA polymerase stalling and

during inﬂammation, such as nitric oxide [39]. The deamination of induction of further repair or damage tolerance mechanisms,

adenosine residues in DNA to inosine has been observed [40]. One leading to mutagenesis.

should note that deamination of ADP and ATP could have led to the Deamination of adenine in DNA results in hypoxanthine, which

formation of IDP and ITP seen in the studies mentioned in the codes as G and thus changes genetic information. When a human c-

preceding paragraphs. Namely, IMP formed from hypoxanthine HA-ras gene containing xanthine was transfected into NIH3T3

could be converted to AMP as shown in Fig. 3, which could then be cells, the presence of xanthine was mutagenic, with a mutation

phosphorylated to ADP and ATP, which in turn could be deaminated frequency of 30–50%, and led to mainly G!A transition mutations,

to IDP and ITP. However, erythrocytes lack adenylosuccinate indicating that thymine was incorporated opposite xanthine

synthetase and thus cannot form AMP from IMP [33]. Nonetheless, [51,52]. Similarly, when hypoxanthine appears in the c-HA-ras

this pathway to ITP is a possibility for the other cell types examined. gene the mutation frequency was 38–50%, with mainly A!G

It is clear that (d)ITP and (d)XTP can be produced in cells, by transition mutations, again consistent with hypoxanthine base

either enzymatic phosphorylation reactions or by deamination of pairing with cytosine [52,53]. Another study in E. coli using an

(d)ATP and (d)GTP. The next logical question is ‘‘What problems oligonucleotide with randomly located inosine residues found that

can be caused by these (d)NTPs that cells need an enzyme to A/T!G/C mutations were formed [54].

remove them?’’ Another possibility is that the presence of hypoxanthine and

xanthine bases in DNA could lead to DNA breaks and chromosomal

4. Problems arising from non-canonical nucleotides instability. To see how this could happen, consider the following

scenario: hypoxanthine or xanthine is recognized as a non-

In the genetic code, only four NTPs are the precursors of RNA standard component of DNA by a DNA repair pathway and is

and likewise only four dNTPs are the precursors of DNA. Thus, the excised, creating a single-strand break (SSB). If a replication fork

presence of (d)NTPs other than these canonical ones could produce encounters this SSB before it is repaired, the fork collapses. When

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P.D. Simone et al. / Mutation Research 753 (2013) 131–146 137

Fig. 4. Problems caused by non-canonical NTPs. If ITP or XTP accumulate in cells, they can be inserted into ribosomal RNA, potentially leading to structural alterations in the

ribosome (indicated by *), or mRNA (indicated by I). Either of these could lead to mistranslation and altered proteins. Alternatively, they can inhibit enzymes that normally use

ATP or GTP. If dITP or dXTP accumulate, they could be inserted into DNA (indicated by I). If not repaired, this could lead to mutations (indicated by *). Alternatively, excision of

the abnormal base creates a SSB, which can lead to DSBs if repair is not completed before replication.

the replication fork from the other direction encounters this and also found in calf thymus [40]. Based on phylogenetic analysis, all

collapses, the result is a double-strand break (DSB) in one of the three domains of life possess a member of the uracil-DNA

daughter chromosomes. This DSB can lead to deletions, transloca- glycosylase superfamily that has hypoxanthine-DNA glycosylase

tions and other forms of chromosomal instability [55–57]. The activity [65]. Lastly, hypoxanthine was found to be released from

enzyme endonuclease V in E. coli recognizes hypoxanthine and inosine-containing DNA incubated with HeLa cell nuclear extracts,

xanthine bases in DNA and nicks the backbone at the second implying the existence of a hypoxanthine-DNA glycosylase in

phosphodiester bond on the 3 end, creating a SSB [49,58,59]. A human cells [37]. The 3-methyladenine-DNA glycosylase ANPG in

similar enzyme exists in rodents and humans, but its enzymatic human cells has been shown to efﬁciently remove hypoxanthine

activity is a subject of debate. Two reports states that it has genuine bases from DNA [66]. The resulting abasic site can then be

EndoV-like speciﬁcity towards inosine [60–62], while another recognized and an AP (apurinic/apyrimidinic) endonuclease or AP

group described its ability to bind branched DNA [63]. E. coli also lyase can initiate repair, opening the DNA backbone and creating a

have a hypoxanthine-DNA glycosylase which releases hypoxan- SSB [67]. This topic will be addressed further in relation to ITPA

thine from DNA, creating an abasic site [64]. A similar enzyme was deﬁciency. ITP has also been identiﬁed as a component of the

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138 P.D. Simone et al. / Mutation Research 753 (2013) 131–146

clastogenic (chromosome breaking) factor found in the plasma of dITP and dXTP accumulate because they are not converted to dIMP

patients with scleroderma [68,69]. and dXMP. The elevated levels of dITP and dXTP lead to their

Lastly, besides the effects from incorporation into DNA or RNA, incorporation into DNA, either during replication or repair. If

ITP and XTP could potentially have negative effects on the endonuclease V is present, it can create a SSB in the process of

numerous proteins that utilize ATP and GTP (Fig. 4, right). Several removing the inosine or xanthosine residue. The SSB can become a

examples of this have been found. For example, the enzyme L- DSB if replication occurs before it is repaired. If RecA is functional,

glutamic acid decarboxylase, involved in the synthesis of the this damage can be repaired, explaining the viability of rdgB single

neurotransmitter g-aminobutyric acid, from rat brain is inhibited mutants. On the other hand, if RecA is absent, the bacteria die due

by ITP [70]. Rabbit psoas muscle ﬁbers did not contract well and to fragmentation of the chromosome and hence rdgB recA double

had disordered striations in the presence of ITP. This is likely due, at mutants are inviable [50]. When nﬁ is additionally mutated, the

least in part, to the slow rate of ITP hydrolysis by actomyosin lethality is suppressed because the SSBs are not created and the

compared to ATP [71]. ITP was also found to inhibit Drosophila DNA chromosome cannot become fragmented. Also, E. coli lacking RdgB

topoisomerase II [72], which could explain, in part, its clastogenic and one of the enzymes that converts IMP to AMP or XMP (i.e.

activity. ITP is known to bind to the F1 subunit of ATP synthase [73], adenylosuccinate synthetase or IMP dehydrogenase, see Fig. 3)

but no studies have shown a detrimental effect. Lastly, ITP and XTP showed large increases in the amount of hypoxanthine bases in the

are both able to activate G-proteins involved in signal transduc- DNA and RNA [45]. These data lend strong support to the ideas

tion. Again, no direct negative effects have been reported, but given presented in previous sections, namely that dITP and dXTP are

the different binding afﬁnities and rates of hydrolysis of GTP, XTP, formed in vivo, that they can be inserted into DNA, and that once in

and ITP, it is possible that these NTPs could function as differential DNA they can have harmful effects.

signal ampliﬁers [74].

The non-canonical nucleotides (d)ITP and (d)XTP can form in 5.2. Yeast

cells. Once formed, they can lead to a variety of problems (Fig. 4).

Thus, the need for an enzyme to remove them is apparent. The ITPA homolog in Saccharomyces cerevisiae was named

Homologs of ITPA have been identiﬁed in all three domains of life. HAM1 [3,75]. When HAM1 was mutated the yeast became

This includes the archaea Methanococcus jannaschii [26] and the supersensitive to the mutagenic effects of the purine analog 6-

bacteria E. coli [49], as well as the eukaryotes Saccharomyces N-hydroxylaminopurine (HAP) [3]. Alignment of the HAM1 protein

cerevisiae [3,75], mice [76], rats [77], rabbits [78], and humans [1]. sequence with the sequence later identiﬁed as human ITPA shows

These facts lead to the conclusion that the function of ITPA must be several regions of high sequence similarity [80]. HAM1 was shown

important. This will be seen by looking at the effect of removing to catalyze the pyrophosphohydrolysis of dITP and dHAPTP, with

ITPA from model organisms. minimal effect on canonical nucleotides [15]. Although HAM1

mutant yeast are hypersensitive to the mutagenic effects of HAP,

5. Effects of ITPA deﬁciency in model organisms the mutant yeast displayed no increase in spontaneous mutation

or recombination [3,75]. This is contrary to what was seen in E. coli,

5.1. E. coli indicating a difference between the two organisms, either in their

degree of dITP/dXTP accumulation or in how they deal with the

The ITPA homolog in E. coli is known as RdgB (Rec-dependent incorporation of non-canonical bases in DNA. It is noteworthy that

growth B). It was originally identiﬁed as being a gene necessary for yeast do not possess an ortholog for Endo V, the enzyme

recombination-defective recA-mutant E. coli to be viable [2]. RdgB responsible for nicking DNA with non-canonical purines in

has (d)NTP pyrophosphohydrolase activity and, like human ITPA, is bacteria. It was proposed that HAP is taken up by yeast, converted

highly speciﬁc for ITP, dITP, and XTP, with minimal activity on to HAP monophosphate by the salvage pathway, and then

canonical (d)NTPs [15,27]. Alignment of the protein sequences of phosphorylated, reduced, and phosphorylated again to eventually

RdgB and ITPA shows 21 strictly conserved residues, many of form dHAPTP. With HAM1 present, this dHAPTP can be detoxiﬁed

which are involved in substrate binding and discrimination or by conversion back to a monophosphate. In the absence of HAM1,

catalysis (see Section 2.2). Lastly, the crystal structure of RdgB dHAPTP can be incorporated into the DNA where it leads to

showed a structure highly homologous to human ITPA mutations [75].

(r.m.s.d. = 1.87 A˚ ). RdgB is therefore a bona ﬁde homolog of ITPA. E. coli and S. cerevisiae are both unicellular organisms and both

What happens when rdgB is mutated and there is a loss of are viable when their ITPA-homolog gene is mutated. Differences in

enzyme activity? Single rdgB mutants are viable and grow the phenotype of the mutants indicate differences between

normally, with normal DNA synthesis. However, the mutant prokaryotes and eukaryotes. What would be the phenotype in a

strains had higher levels of intrachromosomal rearrangements multicellular organism?

than wild type, as well as a greater induction of the SOS response

[2]. The SOS response is a complex pathway in bacteria that is 5.3. Mice

induced by DNA damage and helps the bacteria survive and repair

the DNA. The RecA protein is a key regulator of the SOS response, as In 2009, mice with both alleles of the Itpa gene knocked out

well as being a major player in the repair of DSBs by homologous (Itpa ) were developed [4]. Sakumi et al. reviewed these results

recombination [79]. Hence, E. coli lacking RdgB function seem to so only a brief summary is provided here [47]. Contrary to the

have higher levels of DNA damage, which requires the SOS results in E. coli and yeast, greater than half of the Itpa mice died

response. The greater level of intrachromosomal rearrangements before birth, and those that were born were smaller and died

would suggest that the damage is repaired by some recombination within two weeks. Itpa mice displayed ataxia, abnormal

event mediated by RecA [2]. breathing, immature hair follicles, testicular hypoplasia, and other

Mutating rdgB alone induced DNA damage requiring RecA for abnormalities. The hearts of knockout mice had irregular,

repair, so it makes sense that the rdgB recA double mutant E. coli are asynchronous contractions and they died of cardiac failure. Their

non-viable. What is of interest is the fact that additional mutations hearts were hypoplastic, with thin ventricular walls and disorga-

in the nﬁ gene, which encodes the endonuclease V protein, nized cardiac sarcomeres. Normally the heart and brain express

suppress the lethality of the rdgB recA double mutant [49,59] high levels of ITPA (see Section 2.1) [1,76] and these organs showed

leading to the model presented in Fig. 5. In the absence of RdgB, the most severe phenotype. Rabbit psoas muscle ﬁbers had

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P.D. Simone et al. / Mutation Research 753 (2013) 131–146 139

Fig. 5. E. coli rdgB mutant, synthetic lethality, and rescue. Wild type E. coli do not accumulate dITP or dXTP because RdgB catalyzes their conversion to dIMP and dXMP. In rdgB

mutant E. coli, dITP and dXTP accumulate, leading to their incorporation into DNA (indicated by I/X). Endonuclease V recognizes these lesions and creates SSBs. If the RecA

protein is present, the DNA can be repaired, and so rdgB mutants are viable. If recA is also mutated, then DSBs can accumulate, leading to the inviability of rdgB recA double

mutants. However, mutating the nﬁ gene, which codes for endonuclease V, prevents SSBs from forming and thus rescues the synthetic lethality of rdgB recA double mutants.

Based on [59].

disordered striations and impaired contractions in the presence of study, the same group had identiﬁed human NUDT16 as an enzyme

ITP (Section 4) [71]. This is also consistent with the impaired that binds to ITP. Biochemical characterization of NUDT16 found

/ /

cardiac function of Itpa mice. These observations in Itpa mice that it speciﬁcally hydrolyzes (d)IDP to (d)IMP, with (d)ITP serving

show conclusively that ITPA is serving an important and necessary as a substrate to a lesser extent [82]. Knockdown of NUDT16

role in higher organisms. expression by siRNA reverted the phenotype of immortalized

As further evidence of the fact that ITP can form spontaneously knockout MEFs to that observed in the primary knockout MEFs.

in vivo, Behmanish et al. examined the contents of the erythrocyte The authors proposed that ITPA and NUDT16 have overlapping

nucleotide pool [4]. The Itpa mice had a deﬁnite increase in ITP, roles in protecting cells from (d)ITP. When ITPA is not present,

amounting to 10% of the ATP. Additionally, total heart RNA was NUDT16 could help reduce the levels of (d)ITP by preventing the

found to have elevated inosine (1% of the adenosine level), buildup of (d)IDP, so that it is not further phosphorylated to (d)ITP

providing concrete evidence that ITP can be incorporated into RNA. [81].

If the presence of inosine were due solely to deamination of

adenosine residues already in RNA, then one would not expect to 6. ITPA deﬁciency in humans

see an increase in the knockout mice.

After the Itpa mice were created, primary mouse embryonic 6.1. Human ITPA polymorphism and the Pro32Thr variant

ﬁbroblasts (MEFs) were isolated and characterized [81]. Itpa

MEFs grew signiﬁcantly more slowly and tended to accumulated in Since the role of ITPA in humans cannot be assessed as directly

G2/M phase. Examination of the MEFs for DNA content and as with model organisms, one must rely on ‘‘natural experiments’’

chromosomal abnormalities revealed that the Itpa MEFs had an where genetic changes lead to ITPA deﬁciency. The ﬁrst hint that

increase in ploidy, SSBs, and abnormal chromosomes, particularly ITPA deﬁciency exists in humans came in 1965 when Vanderhei-

ones containing chromatid gaps or breaks. den observed elevated levels of ITP in the erythrocytes of two

The MEFs became spontaneously immortalized after 30–40 siblings [83]. He then examined over 6000 blood samples and

passages and then they appeared like wild type in terms of found seven that had a high ITP concentration [84]. Other studies

proliferation, cell cycle, SSBs, and chromosomal abnormalities. conﬁrmed that low ITPA activity seemed to run in families

NUDT16 protein expression was markedly higher in immortalized [31,32,85]. A major breakthrough came in 2002 when the

knockout MEFs compared to primary knockout MEFs. In a previous underlying genetic cause for ITPA deﬁciency was determined

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140 P.D. Simone et al. / Mutation Research 753 (2013) 131–146

Table 3

this destabilization [10]. The amino acid substitution causes

ITPA polymorphisms associated with decreased activity.

unfavorable steric clashes and simultaneously relaxes the torsional

Genotype Residual Activity Reference constraints of the peptide backbone, with the latter likely being the

most relevant. These lead to large structural changes in the vicinity

94C!A Heterozygote: 22.5% [7,8]

Homozygote: <1% of the mutation, with the major feature being the movement of

IVS2 + 21A!C Heterozygote: 60% [7] Phe31 from the protein’s hydrophobic core out into the solvent

IVS2 + 68T!G Heterozygote: 30% [131]

(Fig. 6). The region around Thr32 and the protein termini become

IVS2 + 68T!C Heterozygote: 60% [132]

a disordered and there is a reduction in secondary structure. The

359_366dupTCAGCACC Heterozygote: 30% [133]

b solvent exposure of Phe31 destabilizes the protein by leaving a

97T!C Heterozygote: 22.8% [119]

cavity in its core and also by causing unfavorable interactions

Table is based on [118].

a between the hydrophobic side chain of Phe31 and water, likely

Duplication of the eight listed residues (359–366).

Leads to a Cys to Arg substitution at amino acid 33. increasing the degree of proteasomal degradation. It was also

proposed that this exposure of Phe31 could lead to Pro32Thr being

directly targeted to the proteasome or sequestered by chaperones.

(Table 3). Several variants in the sequence of the ITPA gene were This is consistent with the observation that human ﬁbroblasts

found [7]. Two were silent and one was in the 3 -untranslated expressing Pro32Thr ITPA have approximately 10-fold lower

region and did not segregate with ITPA activity. A mutation in protein levels than wild type ﬁbroblasts [18].

intron 2 (IVS2 + 21A!C) was found that led to 60% residual ITPA

activity in heterozygotes. The greatest reduction in activity was 6.2. Potential implications of ITPA deﬁciency in human disease

due to a mutation in exon 2 (94C!A), where heterozygotes had

22.5% residual activity and homozygotes had zero ITPA activity. ITPA deﬁciency was initially identiﬁed by the presence of

The activity in compound heterozygotes (94C!A/IVS2 + 21A!C) elevated ITP concentrations in erythrocytes, a seemingly innocu-

was 10%. Another study published later that same year also ous condition [83,84]. On the other hand, overexpression of ITPA

identified the 94C!A mutation as a cause of ITPA deficiency [8]. has been identified in several cancer cell lines (including colon,

The frequency of this allele is highest in Asian populations (11– lung, liver, pancreatic, brain, and others) [90] and MLL-ampliﬁed

15%) and lowest in Central/South American populations (1–2%), acute myeloid leukemia [91]. Likewise, ITPA expression was

with Caucasian and African populations falling in the middle (6– higher in stage III melanoma samples having a poor prognosis

7%) [86]. The Pro32 residue is conserved in mice and Drosophila [1]. compared to samples having a good prognosis, although this

Several other alleles have been identiﬁed that lead to ITPA barely achieved statistical signiﬁcance (P = 0.049) [92]. It should

deﬁciency (see Table 3). No mutations leading to deﬁciency have be pointed out that this overexpression does not necessarily mean

been identiﬁed in the ITPA promoter [87]. that ITPA is a causal factor in cancers. In HeLa cells, inhibition of

Some evidence for how these alleles cause ITPA deﬁciency has ITPA by shRNA leads to sensitivity to HAP-caused DNA breaks and

been found. With the intronic mutations the most obvious apoptosis [93]. However, evidence for a role for ITPA deﬁciency in

hypothesis is that they affect mRNA splicing. Indeed the human disease is mostly indirect and will be brieﬂy summarized

IVS2 + 21A!C mutation leads to reduction in splicing efﬁciency, here.

with the ratio for the C genotype being 80% that of the A genotype The ﬁrst evidence came in 1975, when Fraser and coworkers

[88]. This modest decrease is consistent with the relatively modest found that 16 out of 100 (16%) erythrocyte samples from a

decrease in activity observed. A later study quantiﬁed the amount mentally retarded population accumulated high ITP levels,

of misspliced transcripts for both the IVS2 + 21A!C and the compared to only 4 out of 80 (5%) in a normal population [31].

94C!A alleles [89]. The former led to missplicing of exon 2, while The following year, a study found that the incidence of erythrocyte

the latter led to missplicing of exons 2 and 3. The variant missing ITPA deﬁciency was 2.3 times higher in a psychiatric population

exon 2 was present at 1–5% in wild type individuals, but was versus a normal population. The study included 3477 psychiatric

increased to 16% in an individual homozygous for IVS2 + 21A!C. patients and 6774 normal patients. Within the psychiatric

The variant missing exons 2 and 3 was present at 17% for wild type population, the occurrence of ITPA deﬁciency was greatest among

patients and at 39% and 61% for heterozygotes and homozygotes schizophrenics (particularly paranoid schizophrenics) and a

for 94C!A, respectively. The authors propose that this mutation mentally retarded population. The authors of the study pointed

removes an exonic splicing silencing element, which leads to out that Lesch-Nyhan syndrome, which is characterized by mental

increased missplicing of exons 2 and 3. Clearly, removing an exon retardation and self-mutilation, is caused by hypoxanthine-

from the mRNA would result in a segment of the protein not being guanine phosphoribosyltransferase (HGPRT) deﬁciency. Since

transcribed, producing a nonfunctional enzyme [89]. HGPRT is involved in IMP synthesis (Section 3), it is not

Missplicing accounts for some of the reduced activity seen with unreasonable that deﬁciency of another enzyme involved in

the 94C!A allele, but it does not explain why homozygotes have inosine metabolism, namely ITPA, could lead to psychological

no detectable activity. The 94C!A polymorphism leads to a disorders [94].

Pro32Thr substitution in the protein. This implies that the A follow up study examined ITPA deﬁciency in three groups:

Pro32Thr change in the protein brings about the additional 100 nonpsychiatric patients, 38 chronic paranoid schizophrenic,

reduction in activity. Surprisingly, Pro32Thr ITPA puriﬁed from and 44 chronic undifferentiated schizophrenic. The mean activity

bacteria possessed robust ITPase activity, suggesting that enzy- of ITPA was lower among the schizophrenic population compared

matic deﬁciency in erythrocytes does not stem from the loss of the normal population. Within the schizophrenic group, the

catalysis [18,19]. Given the approximately 25% activity in paranoid schizophrenics had lower ITPA activity. The comparisons

heterozygotes and the fact that the Pro32Thr change would alter achieving statistical signiﬁcance were between normal and all

local secondary structure, it was postulated that the mechanism of schizophrenics, normal and paranoid schizophrenics, and paranoid

catalytic deﬁciency was caused by an impairment of dimer and undifferentiated schizophrenics [95]. As mentioned in section

formation between wild type and variant proteins [7]. This has 4, ITP was shown to inhibit rat brain L-glutamic acid decarboxylase.

been ruled out after it was shown that Pro32Thr ITPA forms a stable Thus, ITPA deﬁciency could lead to ITP build up in the brain, which

dimer, like wild type, but has a lower thermal stability than the could inhibit synthesis of the neurotransmitter g-aminobutyric

wild type enzyme [18]. Structural analysis revealed the cause of acid, possibly producing neurological symptoms [70].

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P.D. Simone et al. / Mutation Research 753 (2013) 131–146 141

Fig. 6. Disruption of the hydrophobic core in Pro32Thr ITPA. (A) The surface of Pro32Thr ITPA shows a hole in the protein structure due to Phe 31 and Thr 32 moving from the

hydrophobic core out into the solvent. (B) An inverse view generated by HOLLOW [135] clearly exhibits the large cavity (gray) left in the protein’s core. The volume of the

cavity is 887 A˚ as determined by VOIDOO [136]. (C) By comparison, the surface of wild type ITPA does not reveal any holes. (D) Only small cavities (gray) exist in the core of

wild type ITPA (volume = 159 A˚ ). Wall-eyed stereo pairs were generated with PyMOL [134].

In contrast, a study by a different group trying to verify these this topic has been done since 1980, leaving the question of

results did not ﬁnd a greater incidence of low ITPA activity among whether or not the results are valid up in the air.

schizophrenic or mentally retarded populations. The study It was recently observed that three out of six patients with

included 30 schizophrenic patients, 35 mentally retarded patients, pulmonary Langerhans’ cell histiocytosis had ITPA deﬁciency,

and a random population of 150 [14]. Unfortunately, no study on which was conﬁrmed by genotyping. This is in contrast to a

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142 P.D. Simone et al. / Mutation Research 753 (2013) 131–146

reference population which only had 11% ITPA deﬁciency. Additional studies found that ITPA 94C!A was associated with

Interestingly, the same three patients with ITPA deﬁciency also early drop-out due to intolerance [102] or leukopenia [103], while

had an unfavorable clinical outcome [96]. Although an intriguing others failed to ﬁnd a signiﬁcant association between ITPA 94C!A

result, further work is needed with larger sample sizes to see if this and adverse reactions [104,105]. A meta-analysis of these studies

association is genuine. If it is, additional work will be required to also did not ﬁnd a signiﬁcant association [106]. Based on these

elucidate the mechanism of how ITPA polymorphisms cause the data, it would seem that the data against an association outweigh

disease. the data for an association.

Lastly, some indirect evidence comes from cell line studies. A Many other studies have come out which lend more support to

human ﬁbroblast cell line homozygous for the 94C!A polymor- the association of ITPA 94C!A with adverse reactions to

phism (producing Pro32Thr ITPA) was examined and found to have mercaptopurines. One found a signiﬁcant association with ﬂu-

a higher level of spontaneous DNA breaks and treatment with HAP like symptoms in an analysis of 207 inﬂammatory bowel disease

induced more breaks than in wild type ﬁbroblasts [9]. So it is patients [107]. Another evaluated children with acute lympho-

possible that the combination of an ITPA polymorphism with a blastic leukemia and showed a higher probability of severe febrile

polymorphism in a DNA repair enzyme could lead to disease. neutropenia in patients with ITPA 94C!A whose mercaptopurine

The fact that ITPA deﬁciency does not seem to produce a severe dosage had been adjusted based on TPMT genotype [108].

phenotype like that seen in Itpa mice deserves consideration. Similarly, a study of Malaysian acute lymphoblastic leukemia

One possibility is that humans express higher levels of NUDT16 patients demonstrated that those with the IVS2 + 21A!C allele

(see Section 5.3), which could help reduce (d)ITP levels [81]. had a greater likelihood of developing fever and liver toxicity [109].

Another likely explanation relies on the fact that the measurement This study is interesting in that it examines an Asian population,

of ITPA activity for the various polymorphisms was done in which has a higher frequency of the ITPA 94C!A allele (Section

erythrocytes. In other cell types, partial loss of ITPA activity could 6.1). Thus, the chance of identifying an association may be higher

be compensated by higher levels of expression. This is supported in an Asian population. A ﬁnal study on acute lymphoblastic

by the observation that although low erythrocyte ITPA activity is leukemia found that a high concentration of 6-methylmercapto-

correlated with lower activity in other cells, the absolute activity in purine nucleotides is associated with hepatotoxicity, and the

these cells is substantially higher than in erythrocytes [85]. Hence, concentration was highest in patients with wild type TPMT alleles

even though 94C!A homozygotes may have zero erythrocyte ITPA and heterozygous for ITPA 94C!A [110]. It may be that ITPA only

activity, the activity in other cells is likely higher and could protect has a role in mercaptopurine toxicity in acute lymphoblastic

from the phenotype observed in knockout mice. leukemia patients. However, a recent study in inﬂammatory bowel

There is some evidence for a direct role of ITPA deﬁciency in disease patients found that low ITPA activity is signiﬁcantly

human disease in that ITPA polymorphism is related to mutagen- associated with adverse reactions, with decreased activities

esis in the mitochondrial genome and blood cancers. A study of accompanied by leukopenia and very low activities accompanied

patients with hematological malignancies found that the 94C!A by increased liver enzymes [111]. It is important to note that this

polymorphism was associated with a signiﬁcantly higher number study looked at ITPA activity rather than a speciﬁc genotype.

of mutation in the mitochondrial DNA (149 mutation in patients The discrepancies between the various studies merit an

carrying the 94C allele vs. 20 mutation in 94A homozygotes). explanation. Stocco et al. suggest that inconsistencies in previous

Additionally, compared to a normal population the frequency of studies may be due to a failure to account for TPMT genotype. Their

the 94C!A polymorphism was higher in patients with myelo- study adjusted mercaptopurine doses based on the TPMT genotype

dysplastic syndrome, although this did not reach statistical of the patients while other studies did not [108]. It could be that the

signiﬁcance [97]. Overall, there is still much to be learned about effects of the TPMT genotype mask the effects due to ITPA when the

the effects of different variants of ITPA in human disease. There is dose is not adjusted. Another possibility is that the low frequency

growing evidence that ITPA deﬁciency is an important pharma- of variant ITPA alleles in some populations makes it difﬁcult to

cogenetic phenotype, leading to either an increase or a decrease in achieve a statistically signiﬁcant result. Lastly, Shipkova and

adverse reactions, depending on the drug used. coworkers propose that the disagreement between studies is likely

due to the fact that most studies only looked for the two most

6.3. ITPA pharmacogenetics common ITPA polymorphisms (94C!A and IVS2 + 21A!C) [111].

Other polymorphisms are known that cause ITPA deﬁciency

ITPA deﬁciency has been found to play a role in negative (Table 3) and there may be others that are not yet known. Variation

responses to several drugs. Marinaki and coworkers were the ﬁrst in activity levels within a given genotype may also play a role.

to discover an association between the 94C!A ITPA variant and Hence, ITPA activity is likely a better metric than ITPA genotype for

increased occurrence of adverse reactions to azathioprine used in assessing the probability of mercaptopurine toxicity.

the treatment of inﬂammatory bowel disease [5,98]. Speciﬁcally, Two other interesting studies have been published regarding

ITPA 94C!A was associated with ﬂu-like symptoms, rash, and ITPA and azathioprine. A case report of a liver transplant recipient

pancreatitis. The IVS2 + 21A!C polymorphism did not predict on azathioprine who presented ﬁfteen years later with nodular

toxicity. Azathioprine and other mercaptopurine drugs are also regenerative hyperplasia found that both the patient and the donor

used in the treatment of leukemias and autoimmune disorders and liver were heterozygous for ITPA 94C!A [112]. This indicates that

are well known to produce negative side-effects such as rash, genotyping of ITPA in both transplant recipients and donor organs

myelotoxicity, pancreatitis, and hepatotoxicity in some patients. may help reduce long-term complications. Other studies demon-

Another enzyme, thiopurine S-methyltransferase (TPMT) also strated that the ITPA 94C!A polymorphism in Japanese popula-

functions in mercaptopurine metabolism and its deﬁciency due tions was associated with an improved response to low-dose

to polymorphism leads to hematopoietic toxicity [99,100]. Many azathioprine in the treatment of systemic lupus erythematosus

studies have been published analyzing the roles of ITPA and TPMT [113,114]. They postulated that accumulation of the metabolite 6-

in mercaptopurine intolerance. Although the contribution of TPMT thioITP could augment the immunosuppression of azathioprine

is well accepted, the results for ITPA have been contradictory. leading to a synergistic effect rather than toxicity at low doses.

Gearry and colleagues examined a larger population of patients A simpliﬁed pathway for the metabolism of mercaptopurines is

with inflammatory bowel disease and failed to find a significant shown in Fig. 7. The active metabolites are the 6-thioguanine

association between ITPA 94C!A and any adverse reactions [101]. nucleotides. 6-thioGTP can be incorporated into DNA and cause

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P.D. Simone et al. / Mutation Research 753 (2013) 131–146 143

Fig. 7. Mercaptopurine metabolism and mechanisms of cytotoxicity. After being taken up into cells, 6-mercaptopurine is converted to 6-thioIMP. (Azathioprine is a prodrug of

6-mercaptopurine.) 6-thioIMP can be converted to 6-thioGMP, which can be phosphorylated to 6-thioGTP. 6-thioGTP is known to cause DNA strand breaks by being

incorporated into DNA and to induce T-cell apoptosis by Rac1 inhibition. 6-thioIMP can also be phosphorylated to 6-thioITP. ITPA can catalyze the conversion of this back to 6-

thioIMP. TPMT can methylate both 6-thioIMP and 6-thioITP. These methylated forms inhibit de novo purine synthesis. In ITPA deﬁcient patients, 6-thioITP and 6-

methylthioITP can accumulate and lead to adverse reactions. Based on [108,116,117].

strand breaks, producing immunosuppression by killing immune [121,122] and others have not [123]. Possible reasons could

cells. This metabolite also inhibits the small GTPase Rac1, leading include different study designs and insufﬁcient sample sizes.

to apoptosis in T-cells. The metabolites 6-methylthioIMP, -IDP, and Another study found no association between the IVS2 + 21A!C

-ITP inhibit de novo purine synthesis [115–117]. ITPA is known to polymorphism and methotrexate efﬁcacy [124]. Given the

be able to catalyze the pyrophosphohydrolysis of 6-thioITP, moderate phenotype associated with this allele the result is not

although with a greater Km and reduced Vmax compared to ITP, surprising. One of the studies also noted an association between

but 6-methylthioITP is a poor substrate [118,119]. It is thought that ITPA 94C!A and gastrointestinal toxicity [122]. The evidence

ITPA deﬁciency leads to accumulation of 6-thioITP and 6- seems to be in favor of ITPA playing a role in methotrexate efﬁcacy,

methylthioITP. This agrees with the increase in 6-methylmercap- but further work needs to be done.

topurine nucleotides in ITPA 94C!A heterozygotes [110]. In wild Lastly, the most recent discovery was the ﬁnding by Felley et al.

type individuals, 6-thioITP can be converted back to 6-thioIMP, that the ITPA deﬁciency alleles 94C!A and IVS2 + 21A!C protect

preventing its accumulation and subsequent methylation by against hemolytic anemia induced by ribavirin used in treating

TPMT. With ITPA deﬁciency, this conversion is severely reduced. hepatitis C [6]. Hemolytic anemia is a treatment-limiting side

A role for ITPA in methotrexate treatment for rheumatoid effect, requiring ribavirin doses to be lowered to less efﬁcacious

arthritis has been examined in several studies. The ﬁrst found an levels. This result has been corroborated by several studies [125–

association between the wild type ITPA gene (the 94C!A 129]. Ribavirin is a nucleoside analog, so it is possible that ITP could

polymorphism was examined) and a good clinical response to compete with ribavirin triphosphate in some process or processes

methotrexate. Methotrexate is believed to help rheumatoid that lead to erythrocyte lysis [6]. However, it has been shown that

arthritis by increasing the release of the anti-inﬂammatory agent ribavirin reduces erythrocyte ATP levels and that ITP can substitute

adenosine. ITPA may inﬂuence the concentrations of AMP and for GTP (which is also reduced by ribavirin) in the adenylosucci-

adenosine by affecting IMP levels, since IMP is a precursor for AMP nate synthetase reaction that is part of the de novo ATP synthesis

(see Section 3 and Fig. 3), providing a possible explanation for why pathway (see Section 3 and Fig. 3). Thus, in ITPA deﬁcient patients,

ITPA deﬁciency is not associated with a good response [120]. As ITP accumulates in the erythrocytes and helps restore ATP levels,

with azathioprine, some reports have conﬁrmed this result preventing lysis due to loss of ATP and the many processes that

Author's personal copy

144 P.D. Simone et al. / Mutation Research 753 (2013) 131–146

encoding inosine triphosphate pyrophosphatase (ITPase), Pharmacogenetics 14

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UNMC Eppley Cancer Center seed grants (Y.I.P. and G.E.O.B.), NCI

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