Recent advances in the pharmacogenomics of thiopurine ... - Nature

The Pharmacogenomics Journal (2001) 1, 254–261

 2001 Nature Publishing Group All rights reserved 1470-269X/01 $15.00

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REVIEW

Recent advances in the pharmacogenomics of thiopurine methyltransferase SA Coulthard AG Hall The Paediatric Leukaemia Research Group, Cancer Research Unit, University of Newcastle Upon Tyne, Newcastle, UK Correspondence: S Coulthard, Paediatric Leukaemia Research Group, Cancer Research Unit, Medical School, Framlington Place, Newcastle Upon Tyne NE2 4HH, UK Tel: ⫹44 0191 222 8947 Fax: ⫹44 0191 222 7556 e-mail: S.A.Coulthard얀ncl.ac.uk

ABSTRACT

The thiopurine drugs (6-mercaptopurine, 6-thioguanine and azathioprine) are commonly used cytotoxic agents and immunosuppressants. One important route for the metabolism of these agents is methylation, mediated by thiopurine methyltransferase (TPMT). It is now well established that interindividual variation in sensitivity to thiopurines can be due to the presence of common genetic polymorphisms affecting the TPMT gene. More recently variations in the number of tandem repeats in the 5⬘ promoter region have been shown to influence TPMT expression in vitro. In this article, we review recent advances in the understanding of the range of inter-individual variation that may be involved in the open reading frame or promoter region of the TPMT gene. We also review the data which have been published regarding the influence such variations may have on both the clinical efficacy and toxicity of the thiopurine drugs. The Pharmacogenomics Journal (2001) 1, 254–261. Keywords: thiopurine methyltransferase; thiopurine; pharmacogenomics; immunosuppressants; leukaemia

INTRODUCTION The fact that individuals may vary dramatically in their response to exposure to specific foods has been recognised for centuries. Pythagoras’ memorable advice to ‘abstain from beans’ may have been based on personal observation of the dramatic haemolytic anaemia that ingestion of fava beans had on fellow Greeks with congenital glucose-6-phosphate dehydrogenase deficiency. Since the introduction of potent medicines, such idiosyncratic responses have also been described following exposure to a variety of drugs. In some cases these responses have an immune basis, for example heparin-induced thrombocytopenia1 but in others they have been shown to be the consequence of variations in the activity of drug metabolising enzymes. With the advent of recombinant DNA techniques the genetic polymorphisms underlying such inter-individual variations have, in a few cases, been described in considerable detail. One of the most extensively studied genes in this regard is TPMT, which encodes thiopurine methyltransferase (EC 2.1.1.67), a key determinant of sensitivity to thiopurine drugs. The aim of this review is to summarise the state of our knowledge of the nature and effect of TPMT polymorphisms, which has, in many ways, led the way in the field of pharmacogenomics.

Received: 9 May 2001 Revised: 3 October 2001 Accepted: 17 October 2001

AN OVERVIEW OF THIOPURINE METABOLISM Three thiopurine drugs are in common usage, and have been for over 40 years (Figure 1). Two, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), form part of many cytotoxic drug regimens for the treatment of acute leukaemia. The third, azathioprine, is a widely used immunosuppressant given to patients with autoimmune conditions, inflammatory bowel disease (Crohn’s) or following kidney

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deoxy-6-thioguanosine 5⬘triphosphate (dTGTP). Incorporation of dTGTP into DNA has been shown to trigger cell cycle arrest and apoptosis by a process that involves the mismatch repair pathway.5 The metabolism of 6-MP to 6-TGMP is less direct than that of 6-TG, involving two additional enzymes: inosine monophosphate dehydrogenase and guanosine monophosphate synthetase. This difference is potentially important as the first intermediate in this pathway, 6-thioinosine 5⬘-monophosphate (tIMP) can act as a substrate for TPMT leading to the production of S-methyl-thioinosine 5⬘-monophosphate (meth-tIMP), a strong inhibitor of purine de novo synthesis (PDNS). Several authors have suggested that PDNS inhibition may make a significant contribution to the cytotoxic action of 6-MP.6–9 In a recent paper Dervieux and colleagues published direct evidence for this hypothesis using CCRFCEM cells transected with the human TPMT gene.10

Figure 1 Chemical structure of the thiopurine drugs.

or heart transplantation. All were designed as purine analogues in order to disrupt normal DNA synthesis and as such are metabolised by the same pathways that control normal nucleotide homeostasis. Azathioprine is rapidly converted to 6-MP and 5⬘substituted 1-methyl-4-nitro-5-thioimidazoles by a process which may involve glutathione and glutathione S-transferases.2,3 The 6-MP moiety is believed to be the main active metabolite although the aminoimidazole may confer additional immunosuppression.4 Both 6-MP and 6-TG are pro-drugs that require activation by hypoxanthine-guanine phosphoribose transferase (HGPRT, Figure 2) prior to the exertion of a cytotoxic effect. Competing with HGPRT for the metabolism of 6-MP are three enzymes, TPMT, aldehyde oxidase and xanthine oxidase (XO). In the case of 6-TG, XO can only metabolise the drug after prior conversion by guanase. The products of these competing reactions produce products with little or no cytotoxic activity. In the case of XO the level of activity in the bone marrow is very low and therefore unlikely to affect the activity of the thiopurine drugs at their main site of action. Metabolism of 6-TG by HGPRT produces 6-thioguanosine 5⬘-monophosphate (6-TGMP), which is further metabolised by a series of kinases and reductases to produce

CLONING AND SEQUENCING OF THE TPMT GENE In most cases the thiopurine drugs are extremely well tolerated, although it is frequently difficult to maintain patients on a stable dose. This is partly due to the fact that the normal route of administration is by mouth and this introduces the variables of compliance and absorption.11,12 However, there is also evidence that there are major inter-individual differences in metabolism. This is now known to be largely due to single nucleotide polymorphisms (SNPs) in the TPMT gene, which affect the catalytic activity and/or protein stability of the enzyme. Weinshilboum and colleagues were the first to report large inter-individual variations in TPMT activity. Using a radiochemical assay to assess red blood cell (RBC) activity they demonstrated a trimodal distribution with approximately 90% of normal individuals having ‘high’ and 10% ‘intermediate’ activity. In one of the 298 cases they studied, TPMT activity was very low.13 Subsequently, using classical segregation analysis of 213 individuals in 49 families they predicted that 66% of the total variance in TPMT activity was controlled by a single gene with a frequency of 0.94 for the allele conferring high activity.14 Lennard et al studied five patients who suffered severe pancytopenia after administration of standard doses of azathioprine and found all of them to have low or undetectable levels of TPMT and abnormally high levels of RBC thioguanine nucleotides.15 Further studies linking clinical response to thiopurines with TPMT genotype have followed the cloning and sequencing of the TPMT gene and its localization to chromosome band 6p22.3.16 The TPMT encodes a 245-amino acid protein with a predicted molecular mass of the 35-kD gene. The genomic structure of the gene has been the subject of some debate. Originally it was reported to be approximately 34 kb in length.16 Later this was modified in a report by Krynetski et al who reported the length to be 25 kb with minor sequence differences.17 Krynetski suggested that the gene has 10 exons and 9 introns but this has been challenged in a paper by Seki et al who could not find the second exon in a study of multiple DNA samples and reported that it was 27 kb in

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Figure 2 Metabolism of 6-TG (a) and 6-TG (b). PRPP, 5⬘-phosphoribosyl-1-pyrophosphate; HGPRT, hypoxanthine guanine phosphoribosyltransferase; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; TPMT, thiopurine methyltransferase.

length18 (GenBank accession number AB045146). Analysis of the 5⬘ promoter region revealed a 71% GC content and no consensus sequences for TATA box or CCAAT elements. More detailed analysis identified stimulating protein 1 (Sp 1) as an important trans-activator essential for constitutive activity in cell culture19 and the presence of a variable number tandem repeat (VNTR) region which may affect levels of expression (see below).20,21 SNPs AFFECTING TPMT ACTIVITY The sequencing of the TPMT gene facilitated detailed studies correlating the presence of SNPs and VNTR length with phenotype. Although studies have been confined to measurements of TPMT activity in RBC, determined by radiochemical assays based on the original method described by Weinshilboum et al,22 a number of studies have been published which demonstrate that this correlates with that in other tissues, including kidney,23 liver24 and leukaemic blasts in children with acute lymphoblastic leukaemia.25 The first SNP in the TPMT gene to be associated with congenital TPMT deficiency and profound 6-MP hypersensitivity was a G238C transversion leading to an alanine to proline substitution at codon 80.26 Heterologous expression of cDNA harbouring the mutation in a yeast system demonstrated a 100-fold reduction in enzyme activity compared to wild-type in spite of normal mRNA levels. This genotype was subsequently designated TPMT*2. Low activity was associated with biallelic transitions in both exons 7 (G460A) and 10 (A719G) leading to substitution of threonine for alanine at position 154 and cysteine for tyrosine at position 240 in four patients with low TPMT activity.16,27 Heterozygotes for this allele, TPMT*3A, were found in patients with intermediate activity. Site-directed mutagenesis was used to create expression constructs containing the G to A transition at position 460 (TPMT*3B) and/or the A to G transition at position 719 (TPMT*3C). Transfection into COS-1 cells showed a decrease in both

The Pharmacogenomics Journal

enzyme activity and immunoreactive protein for all three constructs. In a separate study Tai et al demonstrated that for both TPMT*2 and *3A, low levels of TPMT protein were associated with normal rates of synthesis but a decrease in protein half-life (15 min compared with 18 h for wild-type). Degradation was not impaired if ATP was depleted or if expression was in yeast with mutant proteosomes.28 Since the initial description of TPMT*2 and TPMT*3A, a number of other mutant alleles have been described in patients with intermediate or low TPMT activity including TPMT*3B and TPMT*3C.29–31 With the exception of TPMT*4 which contains a G to A transition which disrupts the intron/exon acceptor splice junction at the final 3⬘ nucleotide of intron 9 these rare alleles contain mutations in the open reading frame of the gene (Figure 3). POPULATION STUDIES The identification of SNPs associated with reduced TPMT activity led to the development of simple restriction fragment length polymorphism assays of polymerase chain reaction products (PCR-RFLP assays) for population studies of relationships between genotype and phenotype. Because of the presence of a processed pseudogene at chromosome band 18q21.1 with an open reading frame homology of 96% compared with TPMT,32 the use of PCR primers traversing intron/exon boundaries has been essential to eliminate false results. Using this approach it has been demonstrated that TPMT*3A mutations are the most common cause of reduced activity in American (allele frequency, 3.2%),33 British (4.5%)34,35 and French (5.7%)36 Caucasians whereas TPMT*3C is more common in African Americans (2.4%),37 Kenyans (5.4%),38 Ghanaians (7.6%),34 Chinese (2.3%)39 and Japanese (1.5%).40 Interestingly in a study of 99 Southwest Asians Collie-Duguid et al could only identify two individuals with a heterozygous mutant allele (TPMT*3) and none with biallelic mutations.39 In the studies reviewed by McLeod et al,29 Ghanaians demonstrated the highest per-


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Figure 3 Thiopurine methyltransferase polymorphisms.1 For deﬁnition of numbering see appropriate reference.2 Single nucleotide polymorphisms responsible for loss of TPMT activity.3 Single nucleotide polymorphisms suspected to be responsible for loss of TPMT activity. Shaded box, untranslated region. Striped box, open reading frame. *The presence of exon 2 has recently been questioned.18

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centage of heterozygotes (14.4%) but in some cases the numbers of subjects included was small and therefore the figures quoted, particularly for patients with biallelic mutations, must be treated with caution. Although the technique of PCR-RFLP is relatively inexpensive and simple to perform, it will only pick up specific SNPs and is not easy to adapt for high-throughput analysis. A variety of techniques including single-strand conformation polymorphism (SSCP) analysis36 and denaturing high performance liquid chromatography41,42 have been used to detect established and novel mutations. In addition an allele-specific fluorogenic 5⬘ nuclease chain reaction assay based on TaqMan technology (Applied Biosystems, Warrington, UK)43 and a multiplexed fluorescence-quenching hybridization probe assay using the LightCycler (Roche) have been described which offer high sensitivity and rapid throughput for clinical and population studies.44 VNTRs IN THE PROMOTOR AFFECTING ACTIVITY As described above, detailed analysis of the promotor region of the TPMT gene by PCR-SSCP revealed inter-individual variations in the 5⬘ flanking region involved in the regulation of TPMT expression.20 Sequence analysis revealed that these variations were due to the presence of 4–8 repeated, 17- or 18-bp units containing putative binding sites for transcription factors. The most frequent alleles contained four or five tandem repeats, a heterozygosity rate of 0.44 was calculated, and a stable Mendelian inheritance of alleles was demonstrated. Modulation of TPMT expression was demonstrated in transient tranfection assays.17 In a subsequent study Yan et al identified repeat units of between 3 and 9 (V3–V9) and confirmed V4 and V5 as the most frequent with frequencies of 0.54 and 0.36 in 279 normal subjects.21,45 VNTR length was compared with RBC TPMT activity. The *V4/*V5 genotype was associated with higher activity than *V4/*V4 or *V5/*V5. Lowest activity levels were associated with genotypes including an allele with more than five repeats. However, in vivo effects were small and did not account for all the variation seen in patients with no detectable open reading frame mutation.20 To date studies have been confined to samples from Caucasians. The experience with SNP analysis suggests that it is important that other ethnic groups are included in future investigations. CLINICAL EFFECTS OF TPMT MUTATIONS Several publications have sought to link TPMT genotype or phenotype with clinical response to thiopurine therapy, either desirable (for example long-term survival in childhood ALL or reduction in kidney transplant rejection episodes) or undesirable (for example neutropenia or hepatotoxicity). In some of these studies the TPMT genotype has been inferred from RBC activity measurements rather than measured directly in at least some of the cases studied. This may give misleading results as TPMT levels have been shown to rise after thiopurine exposure in a number of studies, suggesting either an increase in transcription or a decrease in protein degradation. However, measurements taken before exposure to thiopurines have been shown to


correlate well with genotype.33,35 As noted above, RBC activity correlates with that in other tissues and accurately predicts severe myelosuppression in patients with severe deficiency. In several reports genotype has also been correlated with the level of RBC thiopurine metabolites, including 6-TGNs and methylated mercaptopurine. Thiopurine Induced Myelosuppression There is a clear link between the presence of biallelic mutations and/or low RBC TPMT activity and profound myelosuppression when exposed to standard doses of either azathioprine,15,35,46–49 6-mercaptopurine9,50–51 or 6-thioguanine.52 Studies have also linked 6-MP toxicity with neutropenia in heterozygotes.53 However, in a recent report of 23 patients referred for TPMT assessment because of thiopurine intolerance, only six were deficient and showed biallelic mutations. Four were heterozygotes. Five had intermediate activity but no detectable TPMT*2 or TPMT*3 mutations. The rest had high activity and no detectable mutations.54 These data suggest that factors other than TPMT deficiency can contribute to thiopurine myelotoxicity, supporting observations made by others in patients treated for inflammatory bowel diseases.55,56 Such studies emphasise that, although TPMT genotyping may help to predict severe bone marrow suppression, it remains important to monitor white blood cell counts carefully during the administration of these drugs. Secondary Malignancy In a study of secondary brain tumours in children treated on the Total Therapy Study XII protocol at St Jude Childrens’ Research Hospital, Memphis, USA, Relling et al reported an increased incidence of brain tumours in children who received prophylactic cranial irradiation (six of 52 compared with none of 101 who did not receive radiotherapy).57 Of the six children who developed tumours, four had high RBC TGN levels and three had biallelic TPMT polymorphisms associated with reduced activity. The 8-year cumulative incidence of brain tumour among children with low TPMT activity was 42.9% (SE 20.6) vs 8.3% (SE 4.7; P ⫽ 0.0077). In another study from the same group, there was also a trend for patients treated for ALL who developed secondary AML to have a lower TPMT activity than controls and an association between an earlier onset of secondary AML and lower TPMT activity.58 These conclusions are supported by a study from the Nordic Group who reported a significant association between low TPMT activity and the risk of secondary myelodysplasia or AML.59 Long-Term Survival in Childhood ALL Lennard et al demonstrated an association between RBC TGN levels and probability of relapse in childhood ALL.60 TGN levels were found to be inversely related to RBC-TGN levels but TPMT genotype was unavailable at this time to correlate response directly with the presence of mutant alleles. In a recent study reported by Relling et al, low TPMT activity tended to be associated with a better outcome although this did not achieve statistical significance.61


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Immunosuppression In patients treated with azathioprine to suppress renal allograft rejection the level of increase in TPMT activity was correlated with episodes of graft rejection (34% in those where activity increased, vs 69% amongst those in which it remained unchanged, P ⫽ 0.002).62 These results may indicate a higher level of drug exposure in the patients in which TPMT increased or may indicate that TPMT may increase the intracellular potency of 6-MP. Similar TPMT induction was observed in a study of children treated with azathioprine post renal transplant.63 In this case high levels of TPMT and a significant increase one month after the initiation of azathioprine were related with an increase in the probability of rejection. Further studies are required to resolve these apparently conflicting results. The findings may be confounded by variations in the level of GST mediated conversion of azathioprine into 6-MP. Normal Function of TPMT Completion of sequencing of the human genome has rightly been heralded as a key advance in our understanding of relationships between genotype and phenotype. However, the problem of identifying the normal function of many genes has perhaps been underestimated. In this regard TPMT proves an interesting case in point. Although the sequence of TPMT was identified more than 5 years ago this has not led to the identification of the normal function of the enzyme, which has been studied at the protein level for 40 years. Normal subjects with a congenital absence of TPMT activity do not appear to suffer any ill effects unless and until they are treated with thiopurine drugs. In this regard it is difficult to see how the creation of knock-out mice will assist in the identification of the normal substrate(s) for the enzyme. Studies of the level of TPMT activity in a variety of tissues have shown it to be highly expressed in kidney and liver but as yet detailed immunohistochemical analyses have not been reported to indicate any regional expression within these organs. Genes with homology to TPMT have been found in a variety of organisms including mammals such as cats and dogs, where low levels in cats have been associated with relative azathioprine intolerance,64 and simple organisms such as the pea blight pathogen Pseudomonas syringae pathovar pisi, where overexpression is associated with resistance to tellurite.65 A role for TPMT as a non-specific detoxifying enzyme, analogous to the glutathione S-transferases seems unlikely in view of the limited substrate specificity, relatively high specific activity and low level of expression. The GSTs can act on a wide range of substrates and high levels of expression compensate for their low specific activity. CONCLUSIONS: FUTURE PROSPECTS FOR STUDIES OF TPMT PHARMACOGENETICS In some centres, studies into the role of TPMT in governing response to treatment with the thiopurine drugs has moved from the research laboratory into clinical practice and assessment of RBC TPMT activity has become routine in the investigation of thiopurine associated myelosupression. In a

few instances pretreatment assessment has become mandatory and this has been argued to be cost-effective, at least in the Canadian health care system.66 Elsewhere, physicians have preferred to rely on the monitoring of the white cell count after initiation of therapy.67 However, the onset of neutropenia in patients with biallelic mutations can be very swift and life threatening. It is to be hoped that the increased availability of simple genetic tests and non-isotope based TPMT activity assays will enable the valuable information gained into the role of TPMT mutations in governing drug response to be put into everyday clinical practice. DUALITY OF INTEREST None declared.

ABBREVIATIONS ALL AML dTGTP HGPRT 6-MP meth-tIMP PCR-RFLP PDNS RBC SNPs SSCP Sp1 tIMP 6-TG 6-TGMP TPMT TGNs VNTR XO

acute lymphoblastic leukaemia acute myeloid leukaemia deoxy-6-thioguanosine 5⬘triphosphate hypoxanthine-guanine phosphoribose transferase 6-mercaptopurine S-methyl-thioinosine 5⬘-monophosphate polymerase chain reaction restriction fragment length polymorphism purine de novo synthesis red blood cell single nucleotide polymorphisms single-strand conformation polymorphism stimulating protein 1 6-thioinosine 5⬘-monophosphate 6-thioguanine 6-thioguanosine 5⬘-monophosphate thiopurine methyltransferase thioguanine nucleotides variable number tandem repeat xanthine oxidase

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