Keywords6-mercaptopurine, azathioprine, IMPDH, inflammatory bowel disease, inosine-5′-monophosphate dehydrogenase, thiopurine ---------------------------------------------------------------------- WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Up to 30% of inflammatory bowel disease patients treated with the thiopurine drugs azathioprine and 6-mercaptopurine do not respond properly to therapy. • Genetic variation in the polymorphic enzyme thiopurine S-methyltransferase (TPMT) is associated with adverse events if patients are treated with standard doses.• However, not all adverse events or metabolite patterns can be explained by genetic variations in TPMT, therefore we investigated the role of another thiopurine-metabolizing enzyme, inosine-5′-monophosphate dehydrogenase (IMPDH). WHAT THIS STUDY ADDS• There was a negative correlation of mononuclear cell (MNC) IMPDH activity with red blood cell (RBC) 6-methylthioinosine 5′-monophosphate, but not with RBC 6-thioguanine nucleotide (6-TGN).• The results indicate either that measuring thiopurine metabolites in RBC, as is the current practice in clinical monitoring, is not an appropriate surrogate compartment for MNC metabolite concentrations, or that IMPDH in MNC is not as important a rate-limiting enzyme in the interconversion of thioinosine monophosphate to 6-TGN as has been hypothesized. • All metabolite concentrations and enzymatic activities should preferably be measured in the same compartment. AIMSAzathioprine and 6-mercaptopurine are steroid-sparing drugs used in inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of importance for thiopurine metabolism and occurrence of adverse events. The role of other thiopurinemetabolizing enzymes is less well known. This study investigated the role of inosine-5′-monophosphate dehydrogenase (IMPDH), which is a key enzyme in the de novo synthesis of guanine nucleotides and also strategically positioned in the metabolic pathway of thiopurines. METHODSIMPDH was measured in 100 healthy blood donors. IMPDH, TPMT and metabolite concentrations were studied in 50 patients with IBD on stable thiopurine therapy. IMPDH activity was measured in peripheral blood mononuclear cells. TPMT activity, 6-methylthioinosine 5′-monophosphate (meTIMP) and 6-thioguanine nucleotide (6-TGN) concentrations were measured in red blod cells, which is the current practice in clinical monitoring of thiopurines. Enzyme activities were related to metabolite concentrations and clinical characteristics. RESULTSA wide range of IMPDH activity was observed both in healthy blood donors (median 13.1, range 4.7-24.2 nmol mg -1 protein h -1) and IBD patients (median 14.0, range 7.0-21.7). There was a negative correlation between IMPDH activity and dose-normalized meTIMP concentrations (rs = -0.31, P = 0.03), but no evident correlation to 6-TGN concentration or the meTIMP/6-TGN ratio. There were no significant correlations between TPMT activity and metabolite concentrations. CONCLUSIONEven though the meTIMP concentrations cor...

The polymorphic enzyme thiopurine methyltransferase (TPMT) is involved in the methylation of thiopurines. On comparing the phenotype with the genotype in Swedish patients with inflammatory bowel disease and healthy individuals, we found two discordant cases with low TPMT enzyme activity (0.3 and 0.4 U/ml packed red blood cells (pRBC). Genotyping by pyrosequencing revealed that they carried the nucleotide substitutions 460G>A and 719A>G, giving two possible genotypes (TPMT*1/*3A or TPMT*3B/*3C). DNA sequencing of exon III to X was performed in the patients and their parents. We identified an A>G transition in the start codon (exon III, 1A>G, Met>Val, TPMT*14) in one of the patients and her father (6.3 U/ml pRBC). The mother in this family carried the 460G>A and 719A>G nucleotide substitutions (TPMT*1/*3A; 5.0 U/ml pRBC). In the second family, sequencing revealed a G>A transition in the acceptor splice site in intron VII/exon VIII (IVS7 -1G>A, TPMT*15) in the patient and his mother (6.9 U/ml pRBC). His father was genotyped as TPMT*1/*3A (6.0 U/ml pRBC). Hence, we report the identification of two novel sequence variants, present in highly conserved nucleotide positions of the human TPMT gene, resulting in a loss of enzyme activity.

Background: Interindividual differences in therapeutic efficacy in patients treated with thiopurines might be explained by the presence of thiopurine S-methyltransferase (TPMT) alleles that encode for reduced TPMT enzymatic activity. It is therefore of value to know an individual's inherent capacity to express TPMT. Method: We developed a pyrosequencing method to detect 10 single-nucleotide polymorphisms (SNPs) in TPMT. A Swedish population (n ‫؍‬ 800) was examined for TPMT*3A, TPMT*3B, TPMT*3C, and TPMT*2. Patients with inflammatory bowel disease (n ‫؍‬ 24) and healthy volunteers (n ‫؍‬ 6), selected on the basis of TPMT enzymatic activity, were investigated for all 10 SNPs to determine the relationship between TPMT genotype and phenotype. Results: In the general population we identified the following genotypes with nonfunctional alleles: