Interference free and simplyfied liquid chromatography-based determination of thiopurine S-methyltransferase activity in erythrocytes

The determination of the thiopurine S-methyltransferase activity (TPMT; EC 2.1.1.67) has become an important issue during thiopurine therapy due to its known genetic polymorphism resulting in a wide range of TPMT activity. Therefore, the standard thiopurine drug regimen is associated with increased hematopoetic toxicity in patients with low or absent TPMT activity, whereas patients with high activity may be insufficiently treated. However, presently available methods are labour intensive and time consuming and tend towards too high or too low enzyme activity due to their methodological approach. The use of instable substrate solutions (6-MP or 6-TG), organic solvents like dimethyl sulfoxide and too high substrate and co-substrate saturation concentrations contribute to this phenomenon. We therefore, established an optimized and fast isocratic HPLC linked TPMT assay based on the enzymatic methylation of mercaptopurine or thioguanine in RBC lysates with S-adenosyl-l-methionine as methyl donor. Unspecific non-enzymatic methylation was not detectable. The recovery of 6-methyl-mercaptopurine was 97-102%, the intra- and interday variation between 1.0 and 5.0%, respectively. The assay dispenses with a time consuming extraction procedure with organic solvents, a heating step, and a gradient elution and is therefore, favourable for clinical routine application. The TPMT activity was measured in 62 untreated children with acute lymphoblastic leucemia at the time of diagnosis (activity = 34.0+/-10.6 nmol/g Hb/h, range: 11.5-55.4 nmol/g Hb/h) and in 12 adult healthy volunteers (62.8+/-7.7 nmol/g Hb/h, range: 48-82 nmol/g Hb/h) reflecting the wide measurable TPMT activity found in erythrocytes.     

HPLC/tandem ion trap mass detector methods for determination of inosine monophosphate dehydrogenase (IMPDH) and thiopurine methyltransferase (TPMT)

The efficiency of Mycophenolate mofetil (MMF) and Azathioprine (AZA) as immunosuppressive agents depends on the activity of 2 enzymes, inosine monophosphate dehydrogenase (IMPDH) and thiopurine methyltransferase (TPMT) respectively. We present preliminary evaluation of nonradioactive methods that apply HPLC with ion-trap mass detection to measure the activities of IMPDH in peripheral blood mononuclear cells (PBMC) and TPMT in the erythrocytes (RBC). We found IMPDH activity of 0.9 +/- 0.2 nmol/hour/10(6) PBMC and TPMT activity of 19.9 +/- 4.7 nmol/hour/ml RBC in healthy subjects. These methods, following its further validation, could be useful for monitoring the activity in a clinical and experimental setting.     

Determination of thiopurine S-methyltransferase activity in erythrocytes using 6-thioguanine as substrate and a non-extraction liquid chromatographic technique

A non-extraction high-performance liquid chromatographic (HPLC) method has been developed for the determination of 6-methylthioguanine (6-MTG), as part of the determination of thiopurine S-methyltransferase activity (TPMT) in erythrocytes. Erythrocyte lysate is added to a glass vial containing substrates and incubation buffer, which is then sealed for the rest of the analysis. Enzyme incubation, sample preparation, and analysis are then undertaken without further sample-handling steps. The need for a solvent extraction step has been overcome by heating the incubate to 85 degrees C to stop the enzyme reaction. The heat inactivation step precipitates protein which upon centrifugation forms a thin film in the bottom of the glass vial enabling the supernatant to be injected directly onto the HPLC system. The assay shows excellent precision and recovery with a within-batch imprecision giving a co-efficient of variation of 2.9% (mean=41.5 nmol 6-MTG/gHb/h, n=10) and 5.1% (mean=12.6 nmol 6-MTG/g Hb/h, n=10). The between-batch imprecision gives a co-efficient of variation of 8.2% (mean=11.1 nmol 6-MTG/gHb/h, n=11) and 7.3% (mean=41.0 nmol 6-MTG/gHb/h, n=16). Determination of the TPMT activity in 120 people shows a range of enzyme activity of 11.3-63.8 nmol 6-MTG/gHb/h with a mean and median activity of 34.8 and 34.2 nmol 6-MTG/gHb/h, respectively. TPMT is increasingly used in clinical practice to ensure optimisation of treatment with thioguanine drugs. This direct HPLC method minimises sample-handling, reduces inherent imprecision, the possibility of laboratory error and with the potential for further automation, makes it ideal for use in a regional referral laboratory.     

Determination of thiopurine S-methyltransferase (TPMT) activity by comparing various normalization factors: reference values for Estonian population using HPLC-UV assay

Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) is the key enzyme in the metabolism of thiopurine drugs. Determination of TPMT activity has been used for the individualization of thiopurine dose. We developed HPLC-UV assay for the determination of TPMT activity in human erythrocytes using 6-mercaptopurine as a substrate. Various extraction and chromatographic conditions were compared. In-house developed extraction with acetonitrile provided the lowest limit of quantification. TPMT activity was determined in 99 previously genotyped healthy Estonians. TPMT activity was expressed as the formation of 6-methylmercaptopurine ng/ml/h and normalized either to haemoglobin, haematocrit, erythrocyte count or protein content. The receiver-operating characteristic curve analysis revealed similar accuracy values for TPMT activity in predicting heterozygous and wild type individuals for each method of calculation. In healthy Estonians, TPMT activity varied from 21.5 to 129.6 ng/ml/h. For heterozygous individuals (n = 18), TPMT activity was 48.1 +/- 11.7 ng/ml/h. Wild type individuals (n = 81) revealed significantly higher TPMT activity 79.3 +/- 20.7 ng/ml/h (P < 0.001). This sensitive HPLC assay for quantitative determination of TPMT activity could easily be used in clinical settings. Under constant experimental conditions for haemolysate preparation no normalization is required.     

HPLC/Tandem Ion Trap Mass Detector Methods for Determination of Inosine Monophosphate Dehydrogenase (IMPDH) and Thiopurine Methyltransferase (TPMT)

The efficiency of Mycophenolate mofetil (MMF) and Azathioprine (AZA) as immunosuppressive agents depends on the activity of 2 enzymes, inosine monophosphate dehydrogenase (IMPDH) and thiopurine methyltransferase (TPMT) respectively. We present preliminary evaluation of nonradioactive methods that apply HPLC with ion-trap mass detection to measure the activities of IMPDH in peripheral blood mononuclear cells (PBMC) and TPMT in the erythrocytes (RBC). We found IMPDH activity of 0.9 ± 0.2 nmol/hour/10⁶ PBMC and TPMT activity of 19.9 ± 4.7 nmol/hour/ml RBC in healthy subjects. These methods, following its further validation, could be useful for monitoring the activity in a clinical and experimental setting.     

Thiopurine methyltransferase biochemical genetics: Human lymphocyte activity

The level of human erythrocyte (RBC) thiopurine methyltransferase (TPMT) activity is inherited as a monogenic trait. Experiments were performed to determine whether the level of TPMT activity in the human lymphocyte is regulated in parallel with RBC TPMT. Supernatants of lymphocyte homogenates contained TPMT activity. Lymphocyte TPMT activity was maximal at a reaction pH of 6.6. The apparent K _m value for 6-mercaptopurine, the thiopurine substrate for the reaction, was 8.1×10^–4 m, and the apparent K _m value for S-adenosyl-l-methionine, the methyl donor for the reaction, was 3.6×10^–6 m. The average TPMT activity in lymphocytes isolated from blood of 55 randomly selected subjects was 11.0±0.4 units/10⁹ cells (mean ± SE), with a range of from 4.8 to 17.7 units/10⁹ cells. There was a significant correlation of relative RBC with relative lymphocyte TPMT activity in blood samples from these 55 subjects, with a correlation coefficient of 0.563 (P<0.001). The correlation coefficient for RBC with platelet enyzme activities in these same subjects was also highly significant (r=0.680, P<0.001). Blood samples from four previously identified subjects who were homozygous for the allele TPMT ^L, subjects who lacked detectable RBC enzyme activity, also lacked detectable lymphocyte and platelet TPMT activities. These results were compatible with the conclusion that the genetic polymorphism which regulates RBC TPMT activity also regulates the level of human lymphocyte and platelet TPMT activities.Supported in part by NIH Grants GM 28157 and NS 11014. Dr. Weinshilboum is a Burroughs Wellcome Scholar in Clinical Pharmacology.     

Mercaptopurine pharmacogenetics: Monogenic inheritance of erythrocyte thiopurine methyltransferase activity

Thiopurine methyltransferase (TPMT) catalyzes thiopurine S-methylation, an important metabolic pathway for drugs such as 6-mercaptopurine. Erythrocyte (RBC) TPMT activity was measured in blood samples from 298 randomly selected subjects. Of the subjects, 88.6% were included in a subgroup with high enzyme activity (13.50 ± 1.86 U, mean ± SD), 11.1% were included in a subgroup with intermediate activity (7.20 ± 1.08 U), and 0.3% had undetectable activity. This distribution conforms to Hardy-Weinberg predictions for the autosomal codominant inheritance of a pair of alleles for low and high TPMT activity, TPMT^L and TPMT^H, with gene frequencies of .059 and .941, respectively. If RBC TPMT activity is inherited in an autosomal codominant fashion, then subjects homozygous for TPMT^H would have high enzyme activity, subjects heterozygous for the two alleles would have intermediate activity, and subjects homozygous for TPMT^L would have undetectable activity. The segregation of RBC TPMT activity among 215 first-degree relatives in 50 randomly selected families and among 35 members of two kindreds and one family selected because they included probands with undetectable RBC enzyme activity were also compatible with the autosomal codominant inheritance of RBC TPMT. For example, in eight matings between subjects with intermediate activity (presumed genotype TPMT^L TPMT^H) and subjects with high activity (presumed genotype TPMT^H TPMT^H), 47% (8/17) of the offspring had intermediate activity. This value is very similar to the 50% figure expected on the basis of autosomal codominant inheritance (χ²_[1] = .059). Further experiments are required to determine whether this genetic polymorphism for an important drug metabolizing enzyme may represent one factor in individual variations in sensitivity to thiopurines.     

Thiopurine methyl transferase activity: new extraction conditions for high-performance liquid chromatographic assay

A new liquid–liquid extraction is described for thiopurine methyl transferase (TPMT, EC 2.1.1.67) activity determination: the use of a pH 9.5 NH₄Cl buffer solution, before adding the solvent mixture, allows more rapid extraction, avoiding a centrifugation step, and reduces the global cost of analysis. After the extraction step, 6-methylmercaptopurine, synthesised during the enzymatic reaction, is determined by a liquid chromatographic assay. Analytical performance of the assay was tested on spiked erythrocyte lysates. The linear concentration range was 5–250 ng ml ⁻¹ (r≥0.997, slope=1.497, intercept=−0.367). The recoveries were 82.8, 89.9 and 82.2% for 75, 125 and 225 ng ml⁻¹, respectively. The coefficients of variation were ≤6.1% for within-day assay (n=6) and ≤9.5% for between-day assay precision (n=6; 14 days). TPMT activity was determined in a French adult Caucasian population (n=70). The results ranged from 7.8 to 27.8 nmol h⁻¹ ml⁻¹ packed red blood cells and the frequency distribution histogram is similar to that previously published.     

Human pharmacogenetics of methyl conjugation

Methyl conjugation is an important pathway in drug metabolism. Activities of three human drug-metabolizing methyltransferase enzymes, catechol-O-methyltransferase (COMT) (EC 2.1.1.6), thiopurine methyltransferase ( TPMT ) (EC 2.1.1.67), and thiol methyltransferase (TMT) (EC 2.1.1.9), are controlled by inheritance. COMT activity in the red blood cell (RBC) is regulated by a single genetic locus with two alleles, COMTL for low activity and COMTH for high activity. Gene frequencies of these two alleles were approximately equal in a white population sample of Northern European origin. The genetically controlled level of COMT activity in the RBC reflects the level of enzyme activity in other tissues and is significantly correlated with individual variations in the methyl conjugation of catechol drugs such as L-dopa and methyldopa. TPMT catalyzes the S-methylation of thiopurines and thiopyrimidines . RBC TPMT activity is also controlled by a single genetic locus with two alleles, TPMTL for low and TPMTH for high activity. The gene frequencies of these two alleles were 0.06 and 0.94, respectively, in a white population sample. RBC TPMT activity reflects the level of enzyme activity in other cells and tissues such as the lymphocyte and kidney. TMT catalyzes the S-methylation of aliphatic sulfhydryl compounds such as the drugs captopril and D-penicillamine. The heritability of the level of RBC membrane TMT activity has been estimated on the basis of family studies to be approximately 0.98. Regulation of these three methyl-conjugating enzymes by inheritance raises the possibility that genetically determined methylator status may be one factor responsible for variations in drug metabolism in humans.     

Capillary electrophoresis determination of thiopurine methyl transferase activity in erythrocytes

Thiopurine S-methyltransferase (TPMT) catalyzes methylation of thiopurine drugs (e.g. 6-mercaptopurine, azathioprine). Decreased activity of TPMT is associated with hematopoietic toxicity after administration of standard doses of the drugs. We developed capillary electrophoretic method for determination of TPMT enzyme activity in erythrocytes. Limit of quantification of the method is 1.5 μmol/L (S/N = 6). The recovery of 6-methylmercaptopurine was 87.5–94.8%, imprecision value (as CV, n = 10) was 1.68% (within-day) and 2.53% (between-day). Erythrocyte TPMT activities were measured in 60 healthy adult volunteers.     

The use of denaturing high-pressure liquid chromatography for the detection of mutations in thiopurine methyltransferase

The level of expression of the enzyme thiopurine methyltransferase (TPMT) is an important determinant of the metabolism of drugs used both in the treatment of acute leukaemia (6-mercaptopurine and 6-thioguanine) and as an immunosuppressant in patients with autoimmune diseases or following organ transplantation (azathioprine). Studies of enzyme activity in red blood cells have shown that TPMT expression displays genetic polymorphism with 11% of individuals having intermediate and one in 300 undetectable levels. Patients with biallelic mutations and undetectable enzyme activity suffer life-threatening myelosuppression when treated with conventional doses of these drugs. Patients with intermediate activity have an increased risk of drug-associated toxicity. In the Caucasian populations studied to date, intermediate activity is associated with mutations at two sites of the TPMT gene, G460A and A719G (designated TPMT*3A), in 80% of cases. Detection of these mutations has, to date, been based on the analysis of restriction digests of PCR products. In order to simplify this process we have investigated the ability of denaturing high pressure liquid chromatography (DHPLC) to detect the A719G mutation. DHPLC of PCR products from 15 known heterozygotes (TPMT*3A/TPMT*1) and 18 known homozygotes (TPMT*1/TPMT*1) gave a clear pattern difference between the groups and 100% concordance with the results of restriction digests. These results suggest DHPLC represents a valuable technique for accurate and rapid detection of pharmacologically important mutations in the TPMT gene.     

Thiopurine methyltransferase: a review and a clinical pilot study

Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of 6-mercaptopurine (6MP), which is used in the treatment of acute lymphoblastic leukemia (ALL). TPMT catalyzes the formation of methylthioinosine monophosphate (MetIMP), which is cytotoxic for cultured cell lines, and it plays a role in detoxification of 6MP. Population studies show a genetic polymorphism for TMPT with both high and low activity alleles. About 1 of 300 subjects is homozygous for the low activity. The function TPMT plays in detoxification or therapeutic efficacy of 6MP in vivo is not clear. In this article the genetic polymorphism of TPMT is reviewed and the contribution of TPMT to the cytotoxic action, or detoxification, of 6MP in children with ALL is discussed. Induction of TPMT activity has been described during the treatment for ALL. We performed a pilot study on the influence of high-dose 6MP infusions (1300 mg/m² in 24 h) on TPMT activity of peripheral blood mononuclear cells (pMNC) of eleven patients with ALL. The TPMT activities were in, or, above the normal range. There was no statistically significant difference between the TPMT activities before and after the 6MP infusions. MetIMP levels in pMNC increased during successive courses. This might be explained by TPMT induction, but other explanations are plausible as well. Twenty five percent of the TPMT assays failed, because less than the necessary 5·10⁶ pMNC could be isolated from the blood of leukopenic patients. Red blood cells can not be used for TPMT measurements, since transfusions are frequently required during the treatment with 6MP infusions. Therefore, the influence of high-dose 6MP infusions on TPMT activity can only be investigated further when a TMPT assay which requires less pMNC has been developed.     

Thiopurine methyltransferase activity: new high-performance liquid chromatographic assay conditions

This paper reports change to our previously published high-performance liquid chromatographic method for the measurement of 6-methylmercaptopurine (6-MMP) in red blood cell lysates. The extraction procedure and chromatographic conditions have been improved and the range of the calibration curves has been modified. The recoveries of 10 and 100 ng ml⁻¹ 6-MMP were 99.0±6.0% and 96.3±4.0% respectively and the limit of quantification was lowered to 5 ng ml⁻¹. This method, which does not require radioactive S-adenosyl- -methionine, is more sensitive, specific and reproducible and may prove useful for routine determination of thiopurine methyltranferase activity in red blood cells.     

No induction of thiopurine methyltransferase during thiopurine treatment in inflammatory bowel disease

The aim of this study was to follow, during standardized initiation of thiopurine treatment, thiopurine methyltransferase (TPMT) gene expression and enzyme activity and thiopurine metabolite concentrations, and to study the role of TPMT and ITPA 94C > A polymorphisms for the development of adverse drug reactions. Sixty patients with ulcerative colitis or Crohn's disease were included in this open and prospective multi-center study. Thiopurine na?ve patients were prescribed azathioprine (AZA), patients previously intolerant to AZA received 6-mercaptopurine (6-MP). The patients followed a predetermined dose escalation schedule, reaching target dose at Week 3; 2.5 and 1.25 mg/kg body weight for AZA and 6-MP, respectively. The patients were followed every week during Weeks 1-8 from baseline and then every 4 weeks until 20 weeks. TPMT activity and thiopurine metabolites were determined in erythrocytes, TPMT and ITPA genotypes, and TPMT gene expression were determined in whole blood. One homozygous TPMT-deficient patient was excluded. Five non compliant patients were withdrawn during the first weeks. Twenty-seven patients completed the study per protocol; 27 patients were withdrawn because of adverse events. Sixty-seven percent of the withdrawn patients tolerated thiopurines at a lower dose at Week 20. There was no difference in baseline TPMT enzyme activity between individuals completing the study and those withdrawn for adverse events (p = 0.45). A significant decrease in TPMT gene expression (TPMT/huCYC ratio, p = 0.02) was found, however TPMT enzyme activity did not change. TPMT heterozygous individuals had a lower probability of remaining in the study on the predetermined dose (p = 0.039). The ITPA 94C > A polymorphism was not predictive of adverse events (p = 0.35).     

No Induction of Thiopurine Methyltransferase During Thiopurine Treatment in Inflammatory Bowel Disease

The aim of this study was to follow, during standardized initiation of thiopurine treatment, thiopurine methyltransferase (TPMT) gene expression and enzyme activity and thiopurine metabolite concentrations, and to study the role of TPMT and ITPA 94C > A polymorphisms for the development of adverse drug reactions. Sixty patients with ulcerative colitis or Crohn's disease were included in this open and prospective multi-center study. Thiopurine naïve patients were prescribed azathioprine (AZA), patients previously intolerant to AZA received 6-mercaptopurine (6-MP). The patients followed a predetermined dose escalation schedule, reaching target dose at Week 3; 2.5 and 1.25 mg/kg body weight for AZA and 6-MP, respectively. The patients were followed every week during Weeks 1–8 from baseline and then every 4 weeks until 20 weeks. TPMT activity and thiopurine metabolites were determined in erythrocytes, TPMT and ITPA genotypes, and TPMT gene expression were determined in whole blood. One homozygous TPMT-deficient patient was excluded. Five non compliant patients were withdrawn during the first weeks. Twenty-seven patients completed the study per protocol; 27 patients were withdrawn because of adverse events. Sixty-seven percent of the withdrawn patients tolerated thiopurines at a lower dose at Week 20. There was no difference in baseline TPMT enzyme activity between individuals completing the study and those withdrawn for adverse events (p = 0.45). A significant decrease in TPMT gene expression (TPMT/huCYC ratio, p = 0.02) was found, however TPMT enzyme activity did not change. TPMT heterozygous individuals had a lower probability of remaining in the study on the predetermined dose (p = 0.039). The ITPA 94C > A polymorphism was not predictive of adverse events (p = 0.35).     

Pressure and Temperature Effects on the Activity and Structure of the Catalytic Domain of Human MT1-MMP

Membrane type 1-matrix metalloproteinase (MT1-MMP or MMP-14) is a zinc-transmembrane metalloprotease involved in the degradation of extracellular matrix and tumor invasion. While changes in solvation of MT1-MMP have been recently studied, little is known about the structural and energetic changes associated with MT1-MMP while interacting with substrates. Steady-state kinetic and thermodynamic data (including activation energies and activation volumes) were measured over a wide range of temperatures and pressures by means of a stopped-flow fluorescence technique. Complementary temperature- and pressure-dependent Fourier-transform infrared measurements provided corresponding structural information of the protein. MT1-MMP is stable and active over a wide range of temperatures (10–55°C). A small conformational change was detected at 37°C, which is responsible for the change in activity observed at the same temperature. Pressure decreases the enzymatic activity until complete inactivation occurs at 2 kbar. The inactivation is associated with changes in the rate-limiting step of the reaction caused by additional hydration of the active site upon compression and/or minor conformational changes in the active site region. Based on these data, an energy and volume diagram could be established for the various steps of the enzymatic reaction.     

Genetic analysis of thiopurine methyltransferase polymorphism in a Japanese population

Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathiopurine. Several mutations in the TPMT gene have been identified which correlate with a low activity phenotype. The molecular basis for the genetic polymorphism of TPMT has been established for European Caucasians, African-Americans, Southwest Asians and Chinese, but it remains to be elucidated in Japanese populations. The frequency of the four allelic variants of the TPMT gene, TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3C (A719G) were determined in Japanese samples (n=192) using polymerase chain reaction (PCR)-RFLP and allele-specific PCR-based assays. TPMT*3C was found in 0.8% of the samples (three heterozygotes). The TPMT*2, TPMT*3A and TPMT*3B alleles were not detected in any of the samples analyzed. This study provides the first analysis of TPMT mutant allele frequency in a sample of Japanese population and indicates that TPMT*3C is the most common allele in Japanese subjects.     

The measurement of shear modulus and membrane surface viscosity of RBC membrane with Ektacytometry: a new technique

A new technique is proposed to estimate the shear modulus (mu) and membrane surface viscosity (eta(m)) of red blood cell (RBC). Theoretical formulae for finding these two parameters are first derived based on the force balance on a RBC in a flow field of low viscosity. Different types of Ektacytometry are then used to measure relevant quantities. The obtained values (mu=6.1 x 10(-6)N/m, eta(m)=8.8 x10 (-7)Ns/m for normal RBC) are consistent with those previously found by micropipette technique and in AC electric field. The present technique is, however, much easier to operate and more advantageous in reflecting the average properties of a large quantity of RBCs, and it is much cheaper to be applied in clinical practice than any other method of measuring the two parameters. The sensitivity of the technique is demonstrated by testing RBCs treated with glutaraldehyde of different concentrations. This technique was demonstrated by the flow chamber.     

High-performance liquid chromatographic assay for amiodarone N-deethylation activity in human liver microsomes using solid-phase extraction

A selective and sensitive assay for amiodarone N-deethylation activity in human liver microsomes by high-performance liquid chromatography (HPLC) with UV detection is reported. The extraction of desethylamiodarone from incubation samples was performed by means of an original solid-phase extraction (SPE) procedure using a polymeric reversed-phase sorbent (Oasis HLB). The method was validated for the determination of desethylamiodarone with respect to specificity, linearity, precision, accuracy, recovery, limit of quantitation and stability. Amiodarone N-deethylation activity from low to high substrate concentrations using human liver microsomes was precisely determined without a concentration step. This method is applicable to the study in vitro of the metabolism of amiodarone.     

Comprehensive screening of the thiopurine methyltransferase polymorphisms by denaturing high-performance liquid chromatography

The drug-metabolizing enzyme thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of thiopurines such as 6-mercaptopurine, 6-thioguanine, and azathiopurine, which are used as immunosuppressants and in the treatment of acute lymphoblastic leukemia and rheumatoid arthritis. TPMT enzymatic activity is a polymorphic trait, and poor metabolizers may develop life-threatening bone marrow failure. To avoid such adverse effects, the TPMT enzymatic activity in patients' red blood cells (RBCs) is routinely measured prior to thiopurine administration in a limited number of oncology clinics. In the present study, we took advantage of a highly sensitive and specific automated denaturing high-performance liquid chromatography (dHPLC) technique that not only detects known polymorphic alleles, but also identifies previously uncharacterized sequence variants. We developed a dHPLC-based protocol to analyze the entire coding region and validated the protocol to detect all 16 previously described variant alleles. We further analyzed the entire coding region of the TPMT gene in 288 control samples collected worldwide and identified two novel amino acid substitutions Arg163Cys (487C>T) and Arg226Gln (677G>A) within exons 7 and 10, respectively. The clinical application of this comprehensive screening system for examining the entire TPMT gene would help to identify patients at risk for bone marrow failure prior to 6-mercaptopurine therapy.