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Technical Briefs |
1 Department of Clinical Chemistry, George-August-University, Robert-Koch-Strasse 40, 37099 Göttingen, Germany;
aauthor for correspondence: fax 49-551-39-8551, e-mail nahsen{at}gwdg.de
Inosine triphosphatase (ITPA; EC 3.6.1.19) catalyzes the hydrolysis of ITP to inosine monophosphate, thereby recycling purines that might otherwise be trapped in the form of ITP (1)(2). Two single-nucleotide polymorphisms associated with ITPA deficiency have been identified in the ITPA gene. Individuals who are homozygous for a 94C>A (P32T) mutation have a total deficiency of enzyme activity and accumulate ITP intracellularly, whereas 94C>A heterozygotes have decreased ITPA activity that is 22.5% of the control mean value (2). A second mutation, IVS2 + 21A>C, was detected in ITPA-deficient families. This intronic mutation has a more subtle effect on ITPA activity, and heterozygotes have activities that are, on average, 
60% of the control mean. It was presumed that the IVS2 + 21A>C mutation alters the relatively conserved adenine of a putative splicing branch site, leading to abnormal mRNA splicing (2).
Although ITPA deficiency is not related to any defined pathology in humans, it was recently demonstrated that polymorphisms in the ITPA gene associated with ITPA deficiency have pharmacogenomic implications for patients treated with thiopurines (3). In a retrospective study involving patients with inflammatory bowel disease receiving azathioprine, Marinaki et al. (3) observed that the 94C>A deficient allele was significantly related to the adverse drug reactions (ADRs) flu-like symptoms, rash, and pancreatitis.
The purine analog 6-mercaptopurine and its prodrug azathioprine (AZA) are widely used in the treatment of leukemia and autoimmune disease, and in transplantation. ADRs to these drugs have been related to a genetic deficiency of thiopurine S-methyltransferase (TPMT; EC 2.1.1.67), which is a key enzyme of thiopurine drug catabolism (4). TPMT deficiency leads to life-threatening myelosuppression by accumulation of active thiopurine metabolites (5). Most ADRs to thiopurines, however, cannot be explained by TPMT deficiency. Thiopurines are more frequently discontinued because of non-dose-dependent ADRs (fever, pancreatitis, nausea) than because of dose-dependent side effects (recurrent infections, thrombocytopenia, leukopenia) (6).
In the light of the findings of Marinaki et al. (3), reliable methods are required for screening for the functional polymorphisms in the ITPA gene. We present a procedure for genotyping the ITPA 94C>A and IVS2 + 21A>C point mutations by rapid cycle real-time PCR on the LightCyclerTM (Roche Molecular Biochemicals). We also investigated the molecular basis for the reduced ITPA activity observed with the IVS2 + 21A>C genotype, using a dual-reporter vector system to characterize the splicing efficiencies of the different genotypes.
Primers for amplification of the region of interest in the ITPA gene were located in intron 1 (forward primer; 5'-CTT TAG GAG ATG GGC AGC AG-3') and intron 2 (reverse primer; 5'-CAC AGA AAG TCA GGT CAC AGG-3'). Accumulation of specific PCR product was monitored by use of adjacent hybridization probes designed to bind on one amplicon strand. The 3' end of one probe was labeled with fluorescein (FLU), whereas the 5' end of an adjacent anchor probe was labeled with either Cy5.5 (94C>A) or Bodipy630/650 (IVS2 + 21A>C). Anchor probes were 3'-phosphorylated to prevent probe elongation by the Taq polymerase. All oligonucleotides were synthesized by MWG Biotech. Fluorescence resonance energy transfer occurs when both probes hybridize in close proximity and is detected by the LightCycler. Increasing the temperature during fluorescence reading yields a temperature/fluorescence curve from which the melting point of the probe can be derived. When the appropriate conditions are selected, the mismatch under the detection probe caused by a single-nucleotide polymorphism leads to a substantial decrease in the melting point. Probe and anchor pairs were designed with use of the MeltCalc software (http://www.meltcalc.com), to have a maximum difference in melting temperature (
Tm) between the corresponding genotypes.
The ITPA 94C wild type (wt) was covered by the 3'-FLU-labeled 94Cwt probe (5'-AGT TTC CAT GCA CTT TGG-3') and the adjacent 5'-Cy5.5-labeled 94 anchor probe (5'-GGC ACA GAA AAT TGA CCG TAT GTC TC-3'). The IVS2 + 21C mutation site was detected by the 3'-FLU-labeled IVS2C mut probe (5'-ATG TCT CTG TTT TGT TTT CTT T-3') and a 5'-Bodipy630/650-labeled anchor probe (5'-TAA AAG ATG GTT GGA TTT CTC TGT CTT CCT-3').
PCR reactions were carried out in LightCycler glass capillaries. The reaction mixture consisted of 1 µL of DNA solution, 1 U of native Taq DNA polymerase (PanScript; PAN), 1 µL of 10x PCR buffer (Invitrogen), 0.2 mM each deoxynucleotide triphosphate (Roche Biochemicals), 2.5 mM MgCl2, 500 mg/L bovine serum albumin (New England BioLabs), and 50 mL/L dimethyl sulfoxide (Sigma). Primers were added at a concentration of 0.75 µM, and the probes and anchor probes were added at 0.15 µM. PCR-grade water was added to a final volume of 10 µL.
Each run included a heterozygous DNA control and a water control. The cycling program started with an initial 45 s denaturation step at 95 °C, followed by 35 cycles (95 °C for 0 s, 62 °C for 1 s, and 72 °C for 20 s) with a maximum ramp rate. Melting curve data acquisition in channels 2 and 3 was performed from 30 to 70 °C at a ramp rate of 0.15 °C/s after the last cycle. Melting points of corresponding alleles were determined from six runs as day-to-day imprecision and are shown in Table 1
. Control samples with genotypes ITPA 94 wt/IVS2 + 21 wt and 94C>A/IVS2 + 21 wt and 94 wt/IVS2 + 21A>C were confirmed by sequencing (fmol® DNA Cycle Sequencing System; Promega) on an automated DNA sequencer (Licor).
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The splicing efficiencies of different ITPA genotypes were investigated in a dual-reporter assay. A fragment containing the IVS2 + 21A>C polymorphic site was amplified with PCR primers with attached restriction sites (forward primer, 5'-GTC GAC GTG GCA CAG AAA ATT GAC CG-3'; reverse primer, 5'-GGA TCC CGG CTC CCC CTG GTA CTC C-3', where the underlined bases indicate the SalI and BamHI restriction sites, respectively). Fragments were either IVS2 + 21A or IVS2 + 21C. The restriction sites were used to clone these fragments in frame into the pF2A-GALU plasmid (7). The resulting plasmids contained a ß-gal open reading frame (ORF) without a stop codon, a cloning site, and a luciferase ORF. If an intron with complete splicing signals was cloned between ß-gal and luciferase, the ratio of luciferase to ß-gal activity indicated the splicing efficiency. If inefficient splicing occurs, a stop codon in the retained intron prevents translation of the luciferase ORF. After transfection of HepG2 cells with the plasmids, luciferase and ß-gal activities were measured as described previously (7), and the ratio of luciferase to ß-gal activity was determined.
Typical results for genotyping of ITPA94 and ITPA IVS2 + 21 are shown in Fig. 1A
. Amplification was successful in all 100 anonymous samples from local healthy blood donors. Eighteen blood donors were heterozygous for 94C>A, 22 were heterozygous for IVS2 + 21A>C, and 1 was homozygous for IVS2 + 21A>C. The calculated allele frequencies of 0.09 (94C>A) and 0.12 (IVS2 + 21A>C) for this German Caucasian population are comparable to those reported for a UK Caucasian population (2).
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Screening by real-time PCR represents a fast and reliable method to determine the pharmacogenetic status of a patient with respect to thiopurine treatment. ADRs to these drugs comprise toxic myelosuppression, hepatotoxicity, gastrointestinal disturbances, pancreatitis, and influenza-like symptoms as well as rash. In the majority of patients, the pharmacogenetic basis for side effects is not explained by the TPMT polymorphism (8). There is a well-established link between toxic myelosuppression and the TPMT phenotype (9), but data explaining the other ADRs are conflicting. For example, Schwab et al. (8) demonstrated that gastrointestinal side effects are independent of the TPMT polymorphism, whereas Marinaki et al. (3) described an association of the TPMT genotype with nausea and vomiting. Most recently, in a retrospective study of patients treated with AZA for inflammatory bowel disease, Marinaki et al. (3) revealed a significant association between the 94C>A allele and influenza-like symptoms, rash, pancreatitis, and hepatotoxicity. A significant number of ADRs to thiopurines in patients with normal TPMT activity could therefore be explained by the ITPA polymorphism. Thus, screening for the ITPA genotype allows the prediction of a pharmacogenetic predisposition to ADRs to thiopurines. Marinaki et al. (3) were not able to correlate the IVS2 + 21A>C genotype in their study with AZA toxicity and argued that in these patients the remaining ITPA activity was still sufficient to prevent the accumulation of potentially toxic thio-ITP (3). However, compound heterozygotes (IVS2 + 21A>C and 94C>A) have much lower enzyme activity than 94C>A heterozygotes (2), and such individuals will presumably have an enhanced risk.
Our data indicate a lower splicing efficiency for the IVS2 + 21A>C genotype compared with the wild type. Fig. 1B
shows the reduced ratio luciferase to ß-gal activity, which in the IVS2 + 21C genotype is 80% that of IVS2 + 21A (P = 0.037, paired t-test). The lower splicing efficiency possibly represents the molecular basis for the lower enzyme activity associated with this mutation (2). Previously, Sumi et al. (2) postulated that IVS2 + 21A>C alters a branch point in the ITPA gene. However, because of the distance between IVS2 + 21A>C and the downstream splice site, we presume that the IVS2 + 21A mutation rather alters an extended conserved splice site consensus region next to the exon-intron junction (10).
In conclusion, pretherapeutic ITPA genotyping has the potential to identify patients at increased risk for non-dose-dependent ADRs to thiopurines. Therefore, screening patients for TPMT activity and additionally for ITPA polymorphisms could allow safer and better-tolerated treatment with thiopurines.
Acknowledgments
We gratefully appreciate the technical assistance of Sandra Hartung and Reiner Andag.
References
The following articles in journals at HighWire Press have cited this article:
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  M. Shipkova, K. Lorenz, M. Oellerich, E. Wieland, and N. von Ahsen Measurement of Erythrocyte Inosine Triphosphate Pyrophosphohydrolase (ITPA) Activity by HPLC and Correlation of ITPA Genotype-Phenotype in a Caucasian Population Clin. Chem., February 1, 2006; 52(2): 240 - 247. [Abstract] [Full Text] [PDF]
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N. von Ahsen, V. W. Armstrong, C. Behrens, C. von Tirpitz, A. Stallmach, H. Herfarth, J. Stein, P. Bias, G. Adler, M. Shipkova, et al. Association of Inosine Triphosphatase 94C>A and Thiopurine S-Methyltransferase Deficiency with Adverse Events and Study Drop-Outs under Azathioprine Therapy in a Prospective Crohn Disease Study Clin. Chem., December 1, 2005; 51(12): 2282 - 2288. [Abstract] [Full Text] [PDF]
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, 94C wild type; 
, 94C>A heterozygote; 
, negative control. (Bottom),