IMPDH1 Promoter Mutations in a Patient Exhibiting Azathioprine Resistance

IMPDH1 promoter mutations in a patient exhibiting azathioprine resistance

RL Roberts1, RB Gearry2,3, Around 9% of inflammatory bowel disease (IBD) patients are resistant to 2 1 azathioprine. We hypothesized that these patients may carry mutations ML Barclay and MA Kennedy within inosine-50-monophosphate dehydrogenase (IMPDH). To test this 1Department of Pathology, Christchurch School hypothesis, we screened 20 azathioprine-resistant patients for variations in of Medicine & Health Sciences, Christchurch, the two IMPDH genes (IMPDH1 and IMPDH2) using dHPLC and DNA New Zealand and 2Department of sequencing. A 9 bp insertion within the IMPDH1 P3 promoter was found in a Gastroenterology, Christchurch Hospital, patient exhibiting severe azathioprine resistance. The insertion is predicted to Christchurch, New Zealand abolish a cAMP-response element (CRE) and was found to significantly Correspondence: reduce IMPDH1 P3 promoter activity in a luciferase reporter gene assay Dr RL Roberts, Department of Pathology, (P-value o0.001). This in vitro assay suggests the variant promoter has Christchurch School of Medicine & Health altered function in vivo and consequently may have contributed to the Sciences, P.O. Box 4345, Christchurch, New thiopurine resistance observed in this patient. The absence of functional Zealand. E-mail: [email protected] variants within the other patients indicates that if IMPDH genetic variability contributes to azathioprine resistance it does so infrequently. The Pharmacogenomics Journal (2007) 7, 312–317; doi:10.1038/sj.tpj.6500421; published online 17 October 2006

Keywords: azathioprine resistance; inosine-50-monophosphate dehydrogenase; inflammatory bowel disease; IMPDH1 promoter

Introduction

Azathioprine is an immunosuppressant that undergoes complex metabolism resulting in the generation of several inactive products as well as the active metabolites 6-methylmercaptopurine ribonucleotides (6-MMPR), 6-thio-guanine nucleotides (6-TGN) and 6-thio-inosine triphosphate (6-thio-ITP)1 (Figure 1). The ratio between 6-MMPR and 6-TGN is a major determinant of the efficacy and toxicity of AZA.2–4 A high ratio can result in treatment failure and toxic side effects.3,5–10 Although azathioprine is widely used in the management of inflammatory bowel disease (IBD) approximately 9% of patients are resistant to thiopurine therapy.4,11 These patients are unable to achieve therapeutic concentrations of 6-TGN and accumulate 6-MMPR to hepatotoxic concentrations. Patients are considered to fit the definition of thiopurine resistance if the ratio between 6-MMPR and 6-TGN concentrations within red blood cells (RBCs) exceeds 30:1.11 In most cases azathioprine resistance has been attributed to ultra- 3 Current address: Department of Gastroenter- high thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) activity. However, only ology, Box Hill Hospital, Arnold Street, Box 1–2% of Caucasians exhibit ultra-high TPMT activity.12 As the incidence of Hill, Victoria 3128, Australia thiopurine resistance is far greater than the apparent incidence of ultra-high TPMT activity, we hypothesized that mutations within another purine- Received 25 May 2006; revised 2 August 0 2006; accepted 9 August 2006; published metabolizing enzyme, inosine-5 -monophosphate dehydrogenase (IMPDH, EC online 17 October 2006 1.1.1.205), may be an alternative cause of thiopurine resistance in our IMPDH and azathioprine resistance RL Roberts et al 313

Figure 2a). The P3 promoter variants, including a 9 bp insertion, were found in a single azathioprine-resistant patient with a 6-MMPR:6-TGN ratio of 100:1 (Table 1). All other IMPDH1 variants were either common to both groups or detected solely in azathioprine-responders. Our sample size provided 480% power to detect a statistically significant (two-tailed, a ¼ 0.05) difference in relative risks of between two- and 10-fold for variant rates across these groups (depending on baseline variant rates). Consequently, we are confident that no profound differences in variant rates between azathioprine groups were missed as a consequence of inadequate statistical power. In contrast, the only sequence variation detected in IMPDH2 was a SNP (4999C4T) located in intron 11 (Table 1 and Figure 2b). Of the 13 sequence variants detected in our IBD patients only Figure 1 Metabolism of thioprines. The pro-drug azathioprine (AZA) is the IMPDH1 synonymous SNPs 11401G4C (rs2288550) quickly converted, via a non-enzymatic reaction, to 6-mercaptopurine and15328G4A (rs228075) were among the IMPDH SNPs (6-MP), which undergoes extensive metabolism via three competing pathways, mediated by xanthine oxidase (XO), thiopurine S-methyl- lodged with NCBI dbSNP. To our knowledge, all other transferase (TPMT), and hypoxanthine guanine phosphoribosyl trans- IMPDH variants found in this study have not been ferase (HPRT), respectively. The pathway mediated by XO produces the previously reported in this public database (Table 1). inactive metabolite 6-thiouric acid (6-TU). TPMT catalyses the produc- To assess whether any of the mutations located within the tion of a second inactive metabolite (6-methymercaptopurine, IMPDH1 P3 sequence altered promoter activity, the wild- 1,26,27 6-MMP). The HPRT pathway generates 6-thioinosine monopho- type and variant sequences were cloned into a luciferase sphate (6-TIMP). Once formed, 6-TIMP may be transformed into either reporter vector. The resulting wild-type and variant promo- thioguanine nucleotides (6-TGN) by the rate limiting inosine- ter luciferase constructs exhibited expression levels of 5-monophosphate dehydrogenase (IMPDH) and guanine monopho- 7 7 sphate synthetase (GMPS), methylated into 6-methylmercaptopurine 8.16 0.37 and 6.19 0.01, respectively. The percentage of ribonucleotides (6-MMPR) by TPMT or phosphorylated to 6-thio-inosine maximal expression of the variant promoter relative to the triphosphate (6-thio-ITP). 6-MMPR, 6-TGNs, and 6-thio-ITP are all active wild-type IMPDH promoter across three transfections was metabolites. In normal cells, the accumulation of 6-thio-ITP to toxic 75.9%, indicating that the variant caused a subtle but levels is prevented by inosine triphosphatase (ITPase), which converts 6- significant decrease in IMPDH1 P3 promoter activity in 28 thio-ITP back to 6-TIMP. AZA can act as an immunosuppressant COS-7 cells (non-parametric Mann–Whitney U-test, P-value through the random incorporation of 20-deoxy-6-TGTP into DNA and o0.001). A search of transcription factor binding sites, using RNA.29–31 However, recent research has demonstrated that the primary TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html) way in which AZA exerts its immunosuppressive activity is by inhibiting the guanosine triphosphatase Rac1 in T lymphocytes.32 and the CREB Target Gene database (http://natural.salk.edu/ CREB), found the 9 bp insertion disrupted a putative cAMP-response element (CRE) site within the IMPDH1 P3 promoter (Figure 3). In contrast, the three point mutations population. To test this hypothesis, we screened IBD (À76T4C, À69A4G, and À66A4G) were not predicted to patients who responded to azathioprine and patients who create or abolish a binding site for any known transcription had normal TPMT activity but who were resistant to factor (Figure 3). azathioprine for sequence variations in the two genes Two hundred controls and 100 azathioprine-responsive encoding IMPDH (IMPDH1 and IMPDH2).13,14 IBD patients, who were not included in the initial dHPLC analysis, were genotyped for the P3 promoter variants. None of the four sequence variants were found in either cohort, Results suggesting that this variability within the IMPDH1 P3 promoter is very rare or specific to this IBD patient (Table 1). IBD patients showing resistance to azathioprine had 6- MMPR:6-TGN ratios ranging from 32:1 to 212:1. TPMT enzyme activities of all IBD patients in this study were Discussion between 10.9 and 17.0 IU/ml, indicating none of the azathioprine-resistant patients or responders exhibited ul- Approximately 9% of New Zealand IBD patients on tra-high TPMT activity.9 azathioprine exhibit resistance to this drug.9 It has been Twenty azathioprine-resistant patients and 17 azathio- suggested that ultra-high TPMT enzyme activity is the prine-responders were screened for genetic variability in primary cause of thiopurine resistance,12 however, to our IMPDH using dHPLC. DNA sequencing confirmed the knowledge this has never been proven. In this study, all of presence of 12 IMPDH1 variants, including two synonymous the IBD patients resistant to azathioprine treatment had SNPs (L329L and A525A), one intronic SNP, and four P3 TPMT activities that were well within the normal range for promoter variants, across the 37 IBD patients (Table 1 and our population,9 consequently other factors must be

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Table 1 Allele frequencies of known and novel IMPDH variants found in this study

Variant dbSNP Location Flanking sequencea Allele frequency

dHPLC IBD dHPLC IBD IBD control Non-affected AZA- AZA- populationd control respondersb resistantc (n ¼ 100) population (n ¼ 17) (n ¼ 20) (n ¼ 201)

IMPDH1 À91–83insGAGCAGTAG — Promoter P3 CGGCGT-CAGCAG 0.000 0.025 0.000 0.000 À76T4C — Promoter P3 CAGCAGTAGCAGC 0.000 0.025 0.000 0.000 À69A4G — Promoter P3 AGCAGCAGCAGCA 0.000 0.025 0.000 0.000 À66A4G — Promoter P3 AGCAGCAGCAGCG 0.000 0.025 0.000 0.000 11401G4C (L329L) rs2288550 Exon 7 GCAGCTGCTCTGT 0.210 0.100 — — 14805C4T — Intron 10 GGCAGGCGAGGGC 0.029 0.025 — — 15328G4A (A525A) rs228075 Exon 12 GATCGCGCAGGGT 0.029 0.025 — — 15489G4A — Intron 12 TCTCTCGCTCTTC 0.029 0.000 — — 17493G4A — 3UTR ACCACCGTCCCCA 0.029 0.000 — — 17509T4C — 3UTR GAACCATTCCTCC 0.029 0.000 — — 17529C4T — 3UTR CAGCTGCAGTCGA 0.029 0.000 — — 17535A4C — 3UTR CAGTCGAAGGCTT 0.029 0.000 — —

IMDPH2 4999C4T — Intron 11 CACTGACAGTCTC 0.029 0.000 — — aBase affected by the sequence variant is shown in bold and underlined. bIBD patients with 6-MMP:6-TGN ratios of o30:1. cIBD patients with 6-MMP:6-TGN ratios of 32:1–212:1. dIBD patients with 6-MMP:6-TGN ratios of o30:1 that were not included in the initial dHPLC analysis.

Figure 2 Variability within IMPDH1 and IMPDH2. Light grey rectangles represent exons that encode the open reading frame (ORF); dark grey rectangles represent untranslated regions (UTRs), and promoters are shown as unshaded rectangles. Exons but not introns, are drawn to scale. (a) IMPDH1 is approximately 18 kb, and consists of three alternative promoters (P1, P2 and P3), a 600 bp 50UTR (exons A, B and C), 14 coding exons, and 713 bp of 3’UTR. (b) In contrast, IMPDH2 is only 5.8 kb and although it has an identical ORF structure to IMPDH1, the introns and UTRs of this IMPDH gene are significantly smaller.13 The relative location of variants identified by dHPLC analysis and DNA sequencing are shown with vertical arrows. Numbering of IMPDH1 and IMPDH2 variants is based on the genomic sequences from the UCSC Genome Browser (May 2004 Assembly). In both cases the nucleotide numbered þ 1 is the ‘A’ of the translational initiation codon (ATG) and the nucleotide 50 to þ 1 is numbered À1. responsible for causing thiopurine-resistance in this subset Human IMPDH activity is the result of two separate of patients. It is conceivable that genetic variability in 56 kDa IMPDH enzymes, termed type I and type II, which IMPDH, another purine-metabolizing enzyme, may be an have virtually identical catalytic activities and substrate alternative cause of azathioprine resistance. affinities, and are 84% identical at the amino-acid level.14

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TAG) found in this azathioprine-resistant patient is predicted to disrupt a putative CRE site in the major IMPDH1 promoter. The cAMP-response element-binding protein (CREB) regulates gene expression by binding to a conserved CRE that occurs as either a palindrome (TGACGTCA) or a half site (CGTCA/TGACG).19 A genome-wide analysis of CREB occupancy identified 4084 putative CREB target genes, including IMPDH1.20 Although this insertion, and the point mutations À76T4C, À69A4G and À63A4G, collectively produced a relatively small decrease in IMPDH1 P3 promoter activity in vitro, it suggests the variant promoter has altered function which may be important in vivo. A study of the molecular mechanisms controlling IMPDH2 expression identified CRE sites as an important component of promoter activity in this gene. Mutagenesis of tandem CRE sites reduced IMPDH2 promoter activity by 83% in a transient CAT transfection assay.21 Therefore, it is possible that the disruption of the sole CRE site within the P3 promoter was also sufficient to impair IMPDH1 expression and consequently caused or contributed to the thiopurine resistance observed in our IBD patient. However, the absence of functional variants in the promoter, UTRs and exons of IMPDH1 and IMPDH2 in the other azathioprine-resistant Figure 3 IMPDH1 P3 promoter sequences. The translational initiation patients suggests that if IMPDH genetic variability contri- codon and the sequence immediately 50 of this codon are shown for the butes to thiopurine resistance it does so infrequently. wild-type P3 promoter (a) and the variant P3 promoter (b). The major putative consensus transcription factor binding sites for each sequence, as predicted by TFSEARCH and the CREB Target Gene Database, are Materials and methods indicated by horizontal arrows. The major (M) and minor (m) transcription initiation sites are marked by vertical arrows. Sequence alterations in the variant promoter are indicated in bold lower case and Study participants the wild-type base affected by each point mutation is listed as a capital IBD patients who responded well to azathioprine and those above the variant base. that exhibited resistance to this thiopurine were recruited from gastroenterology clinics at the Christchurch Hospital (Christchurch, New Zealand). Controls living within the Despite the similarities, differences in the regulation and catchment area of Christchurch Hospital were recruited at expression of the two isoforms have been well-documented. random from the electoral roll. Details of the recruitment In general, the level and pattern of IMPDH type I expression and the demographics of this control cohort have been is more variable than that described for the type II isoform.15 described previously.22 Informed consent was obtained from Much of this variability in IMPDH type I expression has all study participants and ethical approval for this research been attributed to the existence of three promoters (P1, P2 was given by the Canterbury Ethics Committee of New and P3) within IMPDH1 that result in the generation of Zealand. All study participants were unrelated and of different mRNA transcripts. Of these, P3 is the major Caucasian ethnicity. promoter producing a 2.5 kb transcript in most tissues.13 IBD patients were defined as azathioprine-resistant if they Furthermore, knockout mouse models have demonstrated had 6-MMPR:6-TGN ratios of 430:1 and no symptomatic that IMPDH type I and type II, in addition to having improvement in IBD despite 44 months azathioprine different levels and patterns of expression, play distinct therapy at 1.5–2.5 mg/kg/day. These patients had sub- physiological roles. For example, a total deficiency in therapeutic 6-TGN concentrations (o235 pmol/8 Â 108 IMPDH type II during the early development of the mouse RBC) and 6-MMPR concentrations exceeding 45700 pmol/ embryo cannot be compensated by IMPDH type I and results 8 Â 108 RBC. They were predicted to have an increased in death of the embryo.16 likelihood of hepatotoxicity and treatment failure due to In this study, we have identified novel mutations within their 6-TGN and 6-MMPR concentrations. In contrast, the IMPDH1 P3 promoter that appeared exclusively in a individuals who experienced disease remission on 1.5– patient exhibiting severe thiopurine resistance. To our 2.5 mg/kg/day were described as being responsive to knowledge, this is the first time that variability in IMPDH1 azathioprine treatment. RBC concentrations of 6-TGN and has been implicated in drug response. All previous muta- 6-MMPR were determined using a previously described tions within this gene have been reported in patients with HPLC method.9 To exclude elevated TPMT enzyme activity autosomal dominant retinitis pigmentosa or leber congeni- as the cause of azathioprine resistance, TPMT activity in tal amaurosis.17,18 The 9 bp insertion (À92–83insGAGCAG RBCs was also determined for each study participant.9

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Screening for variability in IMPDH1 and IMPDH2 (ATCC # CRL-1651) were performed in 24-well plates DNA was collected from the peripheral blood of IBD patients (5 Â 105 cells/well) using Effectene (Qiagen GmbH, Hilden, and controls using guanidium isothiocyanate extraction23 Germany) as per manufacturer’s instructions. To control for and rapid boiling lysis,24 respectively. variation in transfection efficiency among replicates, pro- Twenty azathioprine-resistant and 17 azathioprine- moter constructs were co-transfected with the Renilla responsive IBD patients were screened for mutations within luciferase vector, pRL-TK, (Promega). At 36 h post-transfec- IMPDH1 and IMPDH2 using dHPLC and confirmed by direct tion, COS-7 cells were harvested and firefly and Renilla sequencing. The promoters, open reading frame, and luciferase activities were measured using the Dual-Luciferase untranslated regions (UTRs) of the IMPDH genes were Reporter Assay System (Promega). Luciferase activity was amplified in 45 separate PCR fragments of 200 to 650 bp. calculated as the ratio of firefly to Renilla Luciferase activity. Primers used to amplify the open reading frame of each gene Luciferase values were normalized with COS-7 cells that had were positioned approximately 50–100 bp from each intron– been transfected solely with promoterless pGL3-basic vector. exon boundary to maximize the detection of exonic SNPs Each construct was tested in triplicate across three separate and splice site variants. Primer sequences are available on transfections. request. PCR was carried out in a total volume of 25 ml, containing 200 mM dNTPs, 0.5 mM of each primer, 2.5 mM IMPDH1 P3 promoter genotyping MgCl2, 1 U of Roche Taq Polymerase (Roche Diagnostics IBD patients not included in the initial dHPLC analysis and GmbH, Mannheim, Germany), Roche reaction buffer controls were genotyped for the IMPDH1 P3 promoter B (2.5 ml), and 20 to 100 ng of genomic DNA. The thermal variants. The single base changes (À76T4C, À69A4G cycling conditions were as follows: 2 min at 941C (initial À63A4G) were detected using a dHPLC analysis tempera- denaturation); 35 cycles of 30 s at 941C, 30 s at 581C, 40 s at ture of 681C (44% buffer A, 56% buffer B), whereas a non- 721C; and a final extension of 2 min at 721C. denaturing temperature of 501C was adopted to screen for Denaturing HPLC was performed on a WAVE DNA the 9 bp insertion.25 Fragment Analysis System (Transgenomics Inc., Omaha, NE, USA) equipped with a DNASep column. Unpurified PCR Acknowledgments products were heated to 951C for 5 min and cooled slowly over 45 min to 251C to form heteroduplexes. Six microliters We thank the Canterbury Medical Research Foundation and the Jim of each heteroduplexed PCR product was injected into the & Mary Carney Charitable Trust for their financial support of this DNASep column and separated (flow rate of 0.9 ml/min) study. RLR is the recipient of a NZ S & T post-doctoral fellowship. through a 2% linear acetonitrile gradient. The ratio of Buffer A to Buffer B, and oven temperatures for optimal SNP Duality of Interest detection were pre-determined by WAVEMAKER Software Version 4.1.40. When more than one melting domain was None of the authors have any duality of interest. predicted for an amplicon by the software, the dHPLC analysis was performed at two or more different tempera- References tures. To reduce the probability of missing functionally relevant SNPs that occurred in a homozygous state, each 1 Louis E, Belaiche J. Optimizing treatment with thioguanine derivatives in amplicon derived from a case was heteroduplexed with an inflammatory bowel disease. Best Pract Res Clin Gastroenterol 2003; 17: amplicon known from DNA sequencing to be identical to 37–46. the IMPDH1 and IMPDH2 mRNA reference sequences 2 Hindorf U, Lyrenas E, Nilsson A, Schmiegelow K. 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