Intermediate Thiopurine S-methyltransferase Activity Research Articles

Thiopurine S-methyltransferase (TPMT) Activity Is Better Determined by Biochemical Assay Versus Genotyping in the Jewish Population

Thiopurine S-methyltransferase (TPMT) is a key enzyme that deactivates thiopurines, into their inactive metabolite, 6-methylmercaptopurine. Intermediate and low TPMT activity may lead to leukopenia following thiopurine treatment. The aim of this study was to determine TPMT activity and TPMT alleles (genotype-phenotype correlation) in Jews, aiming to develop an evidence-based pharmacogenetic assay for this population. TPMT activity was determined in 228 Jewish volunteers by high performance liquid chromatography. Common allelic variants in the Caucasian population [TPMT*2 (G238C), TPMT *3A (G460A and A719G), TPMT* 3B (G460A) and TPMT*3C (A719G)] were tested. Phenotype-genotype correlation was examined and discordant cases were fully sequenced to identify novel genetic variants. Mean TPMT activity was 15.4 ± 4 U/ml red blood cells (range 1-34). Intermediate activity was found in 33/228 (14%) subjects and absent activity was found in one sample (0.4%). Only eight individuals (3.5% of the entire cohort and 24% of those with intermediate/low activity) were identified as carriers of a TPMT genetic variant, all of whom had the TPMT*3A allele. Sequencing the entire TPMT coding region and splice junctions in the remainder of the discordant cases did not reveal any novel variants. Genotyping TPMT in Jews yields a much lower rate of variants than identified in the general Caucasian population. We conclude that a biochemical assay to determine TPMT enzymatic activity should be performed in Jews before starting thiopurine treatment in order to identify low activity subjects.

OC-015 The Influence of Gender and Haemoglobin on TPMT Activity

IntroductionPre-treatment measurement of red blood cell (RBC) thiopurine-S-methyltransferase (TPMT) activity is recommended to guide initial dosing of azathioprine (AZA) and mercaptopurine (MP). TPMT exhibits a trimodal distribution, with low and...

Genotyping should be considered the primary choice for pre-treatment evaluation of thiopurine methyltransferase function

Background and aimsA pre-treatment determination of the thiopurine S-methyltransferase (TPMT) genotype or phenotype can identify patients at risk of developing severe adverse reactions from thiopurine treatment. The risk of misclassifying a patient might be dependent on the method used. The aim of this study was to investigate the concordance between TPMT genotyping and phenotyping. MethodsThe data consist of 7195 unselected and consecutive TPMT genotype and phenotype determinations sent to the division of Clinical Pharmacology, Linköping, Sweden. TPMT activity was measured in red blood cells (RBC) and the genotype determined by pyrosequencing for the three most common TPMT variants (TPMT *2, *3A, *3C). ResultsTPMT genotyping identified 89% as TPMT wild type (*1/*1), 10% as TPMT heterozygous and 0.5% as TMPT defective. The overall concordance between genotyping and phenotyping was 95%, while it was 96% among IBD patients (n=4024). Genotyping would have misclassified 8% of the TPMT defectives as heterozygous as compared to 11% if only TPMT activity had been measured. 11% of the heterozygous patients had a normal TPMT activity (>8.9U/ml RBC) and 3% of the TPMT wild-type patients had an intermediate TPMT activity (2.5–8.9U/ml RBC). ConclusionsThere is a risk for TPMT misclassification when only genotyping or phenotyping is used, but it is not reasonable to check both in all patients. Since TPMT genotyping is the more reliable test, especially in TPMT heterozygotes, we suggest that genotyping should be considered the primary choice for the pre-treatment evaluation of TPMT function before initiation of thiopurine therapy.

Are Patients with Intermediate TPMT Activity at Increased Risk of Myelosuppression When Taking Thiopurine Medications?

Thiopurine S-methyltransferase (TPMT) metabolizes thiopurine medications, including azathioprine and 6-mercaptopurine. Absent TPMT activity (i.e., in individuals homozygous for a variant TPMT allele) is associated with an increased risk of myelosuppression in patients taking thiopurine drugs. However, it is not clear if there is also an increased risk for patients with intermediate TPMT activity (i.e., in individuals heterozygous for a variant TPMT allele). To quantify the increased risk of myelosuppression for patients with intermediate TPMT activity. A systematic review identified published studies, up to 29 September 2008, that explored the relationship between TMPT and hematological adverse drug reactions to thiopurines. Following a critical appraisal of the quality of published studies, a meta-analysis calculated the odds ratio of myelosuppression for patients with intermediate TPMT activity compared with wild-type. A total of 67 studies were identified, the majority retrospective cohort in design. Patients with two TPMT variant alleles who are TPMT deficient have a substantial increase in their risk of myelotoxicity (86% of deficient patients developed myelosuppression). The increase in odds ratio of developing leukopenia for patients with intermediate TPMT activity or one TPMT variant allele compared with wild-type was 4.19 (95% CI: 3.20-5.48). This meta-analysis suggests that individuals with both intermediate and absent TPMT activity have an increased risk of developing thiopurine-induced myelosuppression, compared with individuals with normal activity. However, there is significant variability in the quality of the reported studies and large prospective studies to clarify the size of the effect of TPMT variant alleles on the risk of myelosuppression should be conducted. Accurate risk assessments will provide important data to inform clinical guidelines.

Thiopurine S-methyltransferase alleles, TPMT*2, *3B and *3C, and genotype frequencies in an Indian population

Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds including thiopurine drugs such as 6-mercaptopurine, 6-thioguanine and azathioprine. TPMT activity exhibits genetic variation and shows tri-modal distribution with 89-94% of individuals possessing high activity, 6-11% intermediate activity and approximately 0.3% low activity. Patients with intermediate or deficient TPMT activity exposed to thiopurine drugs show severe hematopoietic toxicity. Three single nucleotide polymorphisms (SNPs) in TPMT (NM_000367.2:c.238G>C, NM_000367.2:c.460G>A and NM_000367.2:c.719A>G) define the most prevalent mutant alleles associated with loss of catalytic activity reported in several populations. The present study investigated, for the first time, the frequency distribution of these three SNPs of TPMT, their alleles and genotypes in a Southern Indian population. Peripheral blood was obtained from 326 individuals of a Southern Indian population, and genomic DNA was isolated from total peripheral white blood cells. The genotypes at the polymorphic loci were determined by allele-specific polymerase chain reaction, restriction fragment length polymorphism and confirmatory DNA sequencing. The estimated genotype frequency for homozygous TPMT(*)1/(*)1 was 97.24%, for heterozygous TPMT(*)1/(*)2 and TPMT(*)1/(*)3B, 0.61% each, and for heterozygous TPMT(*)1/(*)3C, 1.53%. The frequency of heterozygous mutants in the studied Indian population was 2.76%. This study demonstrated significant variations in TPMT gene polymorphisms in an Indian population in relation to other human populations and may help to predict both clinical efficacy and drug toxicity of thiopurine drugs.

Relationships between thiopurine S-methyltransferase polymorphism and azathioprine-related adverse drug reactions in Chinese renal transplant recipients

To systematically investigate the relationships between thiopurine S-methyltransferase (TPMT) polymorphisms and azathioprine-related adverse drug reactions in patients with kidney transplantation. Erythrocyte TPMT activity of 150 patients with kidney transplantation and AZA therapy was determined by HPLC. The frequency of four common TPMT mutant alleles, TPMT*2, *3A, *3B, and *3C was determined by allele-specific PCR and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. Thirty cases (20%) had stopped azathioprine medication or were on reduced dose due to azathioprine-related side effects. The TPMT activity range of cases who never experienced side effects was 16.63-68.25 U, the mean of the controls was 38.43 +/- 11.59 U. The mean value of 12 cases with hematotoxicity was 23.50 +/- 10.33 U, much lower than the control mean (P < 0.05). No significant difference between the mean value of 18 cases with hepatotoxicity and the control mean (P > 0.05) was seen. No case with TPMT deficiency was found in all patients studied, and TPMT*2, *3A, and *3B were not detected in any of them. TPMT*3C heterozygous alleles were found in 4.7% (seven cases) of these patients, all seven cases had intermediate TPMT activity, and the mean was 16.75 +/- 2.09 U, much lower than other TPMT wild-type patients (P < 0.05). In the seven TPMT*3C patients, four cases experienced side effects (hematotoxicity, n = 2; hepatotoxicity, n = 2). This study demonstrates that TPMT activity is reduced in patients with TPMT*3C mutation. AZA-induced hematotoxicity is related to the reduced TPMT activity.

Thiopurine S-methyltransferase (TPMT) pharmacogenetics: three new mutations and haplotype analysis in the Estonian population

Thiopurine methyltransferase (TPMT) is a cytoplasmic enzyme involved in the metabolism of thiopurine drugs. To date, at least 25 single nucleotide polymorphisms have been reported in the TPMT gene, 23 of these are associated with reduced enzyme activity. The aim of the present study was to sequence the whole coding region of TPMT (exons 3-10) to identify known and novel TPMT sequence variants amongst healthy Estonians. Erythrocyte TPMT activity was also measured to carry out a genotype-phenotype comparison. A total of 21 subjects were heterozygous for known TPMT alleles (*2, *3A, *3C, *9, *12). Several other previously described intronic and exon polymorphisms were identified. Three novel mutations were detected -30T>A in exon 3, 10A>G in intron 3, and 145A>G in intron 10. Association analysis revealed four markers (114T>A, 94T>A, 460G>A, 719A>G) whose frequencies were significantly different in intermediate (enzyme activity <or=60 ng/mL/h) methylators compared to normal (enzyme activity 61-139 ng/mL/h) and high (enzyme activity >or=140 ng/mL/h) methylators (p<0.001). Haplotype analysis found one haplotype to be associated with intermediate TPMT activity. Our results point to several markers that predict reduced enzyme activity. None of the identified markers were associated with high enzyme activity.

TPMT genotype and its clinical implication in renal transplant recipients with azathioprine treatment

Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. Patients with intermediate or deficient TPMT activity are at risk of toxicity after receiving standard doses of these drugs. This study determined the frequencies of TPMT alleles (TPMT*2, *3A, *3B and *3C) and explored the association between TPMT genetic polymorphism and the development of adverse drug reactions in Chinese renal transplant patients receiving azathioprine (AZA). TPMT genotypes were determined using polymerase chain reaction-based assays in 122 renal transplant patients and 210 healthy subjects. Biochemical and clinical data were retrospectively evaluated after renal transplantation. Of 122 patients, eight (allele frequency 3.28%) were heterozygous for TPMT*3C and no TPMT*2, *3A or *3B or homozygous TPMT*3C subjects were identified. The pattern and frequency of the main mutant TPMT alleles were similar in patients and healthy subjects. Four of five patients (80%) with haematopoietic toxicity were heterozygotes. TPMT heterozygosity was associated with significant reductions in haematological indices and a significant decrease in cyclosporine plasma concentrations in the first year after renal transplantation. No association between TPMT genotype and renal rejection was identified. Our results, together with those of others pointing in the same direction, suggest that genotyping the major TPMT variant alleles may be a valuable tool in preventing AZA toxicity and optimization of immunosuppressive therapy.

Thiopurine methyltransferase Genotype Is Not a Risk Factor for Secondary Malignant Neoplasias after Treatment for Childhood Acute Lymphoblastic Leukemia on Berlin-Frankfurt-Muenster Protocols.

The thiopurines mercaptopurine and thioguanine are important components of contemporary polychemotherapeutic treatment protocols for acute leukemias. Thiopurines are prodrugs that undergo intestinal and hepatic metabolism. Activation occurs via a multistep pathway to form thioguanine nucleotides, which are thought to be the major cytotoxic compound through triggering cell cycle arrest and apoptosis. This process is in competition with direct inactivation of thiopurines or their metabolites by thiopurine S-methyltransferase (TPMT). TPMT is a cytosolic enzyme that is ubiquitously expressed in the human body and catalyzes the S-methylation of thiopurines. The TPMT locus is subject to genetic polymorphism, with heterozygous individuals (5 to 11% of Caucasians) having intermediate TPMT activity, and homozygous individuals (0.3 to 0.5% of Caucasians) having low TPMT activity. At least 20 variant TPMT alleles (*2 to *18) have been described so far that confer decreased enzyme activities compared to the TPMT*1 wild-type allele. TPMT genotype is highly concordant with TPMT phenotype. With regard to long-term adverse effects, patients who have diminished TPMT activity were shown to be at increased risk of developing chemotherapy-induced acute myeloid leukemia and radiation-induced second brain tumors tumors after exposure towards mercaptopurine during therapy for childhood acute lymphoblastic leukemia (ALL). To investigate if such an association is generalizable to other entities of secondary malignant neoplasms (SMN) and different treatment approaches for ALL, we collected specimens of 72 patients who developed a SMN after ALL treatment on Berlin-Frankfurt-Muenster (BFM) protocols, analyzed their TPMT genotype and compared genotype frequencies to these in the general ALL patient population. The 72 patient cohort consisted of 49 hematological SMN (half of them received cranial irradiation), 12 brain tumors, and 11 other solid SMN. Neither in the entire patient group nor in subgroup analyses, differences in allele frequencies of TPMT variants conferring diminished enzyme activity were detectable when comparing SMN patients to the overall ALL patient population. Thus, low TPMT activity does not seem to play a major role in the etiology of SMN after treatment for childhood ALL according to BFM treatment strategies. Factors potentially helpful for the explanation of the previously described relationsship of TPMT activity with SMN will be presented.

Frequency Distribution of Thiopurine S-Methyltransferase Alleles in a Polish Population

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. TPMT activity exhibits an interindividual variability mainly a result of genetic polymorphism. Patients with intermediate or deficient TPMT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. It has previously been reported that 3 variant alleles:TPMT*2, *3A, and *3C are responsible for over 95% cases of lower enzyme activity. The purpose of this study was to determine the frequency of TPMT variant alleles in a Polish population. DNA samples were obtained from 358 unrelated healthy Polish subjects of white origin, and TPMT genetic polymorphism was determined using PCR-RFLP and allele-specific PCR methods. The results showed that allelic frequencies were 0.4% for TPMT*2, 2.7% for TPMT*3A, and 0.1% for TPMT*3C, respectively. A TPMT*3B allele was not found in the studied population. The general pattern of TPMT allele disposition in the Polish population is similar to those determined for other white populations, but the frequency of total variant alleles is lower than in other European populations studied to date.

Comprehensive analysis of thiopurine S-methyltransferase phenotype-genotype correlation in a large population of German-Caucasians and identification of novel TPMT variants.

The thiopurine S-methyltransferase (TPMT) genetic polymorphism has a significant clinical impact on the toxicity of thiopurine drugs. It has been proposed that the identification of patients who are at high risk for developing toxicity on the basis of genotyping could be used to individualize drug treatment. In the present study, phenotype-genotype correlation of 1214 healthy blood donors was investigated to determine the accuracy of genotyping for correct prediction of different TPMT phenotypes. In addition, the influence of gender, age, nicotine and caffeine intake was examined. TPMT red blood cell activity was measured in all samples and genotype was determined for the TPMT alleles *2 and *3. Discordant cases between phenotype and genotype were systematically sequenced. A clearly defined trimodal frequency distribution of TPMT activity was found with 0.6% deficient, 9.9% intermediate and 89.5% normal to high methylators. The frequencies of the mutant alleles were 4.4% (*3A), 0.4% (*3C) and 0.2% (*2). All seven TPMT deficient subjects were homozygous or compound heterozygous carriers for these alleles. In 17 individuals with intermediate TPMT activity discordant to TPMT genotype, four novel variants were identified leading to amino acid changes (K119T, Q42E, R163H, G71R). Taking these new variants into consideration, the overall concordance rate between TPMT genetics and phenotypes was 98.4%. Specificity, sensitivity and the positive and negative predictive power of the genotyping test were estimated to be higher than 90%. Thus, the results of this study provide a solid basis to predict TPMT phenotype in a Northern European Caucasian population by molecular diagnostics.

Pyrosequencing of TPMT alleles in a general Swedish population and in patients with inflammatory bowel disease.

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. 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. In the general population we identified the following genotypes with nonfunctional alleles: TPMT*1/*3A (*3A allelic frequency, 3.75%), TPMT*1/*3C (*3C allelic frequency, 0.44%), TPMT*1/*3B (*3B allelic frequency, 0.13%), and TPMT*1/*2 (*2 allelic frequency, 0.06%). All nine individuals with normal enzymatic activity were wild-type TPMT*1/*1. Thirteen individuals with intermediate activity were either TPMT*1/*3A (n = 12) or TPMT*1/*2 (n = 1). Eight individuals with low enzymatic activity were TPMT*3A/*3A (n = 4), TPMT*3A/*3C (n = 2), or TPMT*1/*3A (n = 2). Next to wild type, the most frequent alleles in Sweden are TPMT*3A and TPMT*3C. A previously established phenotypic cutoff for distinguishing normal from intermediate metabolizers was confirmed. To identify the majority of cases (90%) with low or intermediate TPMT activity, it was sufficient to analyze individuals for only 3 of the 10 SNPs investigated. Nevertheless, this investigation indicates that other mutations might be of relevance for decreased enzymatic activity.

Molecular analysis of thiopurine S-methyltransferase alleles in South-east Asian populations.

Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. In Caucasians, four variant TPMT alleles have been detected in over 80% of individuals with low or intermediate TPMT activity. The wild-type allele is designated as TPMT*1 and the mutant alleles are designated TPMT*2 through TPMT*8. The frequency of these alleles in different ethnic groups has not been well defined. In this study, one hundred individuals, from each of the Indonesian, Thai and Philippine populations, together with 249 Taiwanese, were analysed by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing methods. The results showed that the allelic frequencies of TPMT*3C were 0.6% for Taiwanese and 1% for Filipino, Thai and Indonesian populations, respectively. One Filipino with a Caucasian parent was found to be heterozygous for the TPMT*2 allele. This study provides the first analysis of the allele frequency distribution of all known TPMT mutations in South-east Asian populations.

Preponderance of thiopurine S-methyltransferase deficiency and heterozygosity among patients intolerant to mercaptopurine or azathioprine.

To assess thiopurine S-methyltransferase (TPMT) phenotype and genotype in patients who were intolerant to treatment with mercaptopurine (MP) or azathioprine (AZA), and to evaluate their clinical management. TPMT phenotype and thiopurine metabolism were assessed in all patients referred between 1994 and 1999 for evaluation of excessive toxicity while receiving MP or AZA. TPMT activity was measured by radiochemical analysis, TPMT genotype was determined by mutation-specific polymerase chain reaction restriction fragment length polymorphism analyses for the TPMT*2, *3A, *3B, and *3C alleles, and thiopurine metabolites were measured by high-performance liquid chromatography. Of 23 patients evaluated, six had TPMT deficiency (activity < 5 U/mL of packed RBCs [pRBCs]; homozygous mutant), nine had intermediate TPMT activity (5 to 13 U/mL of pRBCs; heterozygotes), and eight had high TPMT activity (> 13.5 U/mL of pRBCs; homozygous wildtype). The 65.2% frequency of TPMT-deficient and heterozygous individuals among these toxic patients is significantly greater than the expected 10% frequency in the general population (P <.001, chi(2)). TPMT phenotype and genotype were concordant in all TPMT-deficient and all homozygous-wildtype patients, whereas five patients with heterozygous phenotypes did not have a TPMT mutation detected. Before thiopurine dosage adjustments, TPMT-deficient patients experienced more frequent hospitalization, more platelet transfusions, and more missed doses of chemotherapy. Hematologic toxicity occurred in more than 90% of patients, whereas hepatotoxicity occurred in six patients (26%). Both patients who presented with only hepatic toxicity had a homozygous-wildtype TPMT phenotype. After adjustment of thiopurine dosages, the TPMT-deficient and heterozygous patients tolerated therapy without acute toxicity. There is a significant (> six-fold) overrepresentation of TPMT deficiency or heterozygosity among patients developing dose-limiting hematopoietic toxicity from therapy containing thiopurines. However, with appropriate dosage adjustments, TPMT-deficient and heterozygous patients can be treated with thiopurines, without acute dose-limiting toxicity.

High-Throughput Genotyping of Thiopurine S-Methyltransferase by Denaturing HPLC

The thiopurine S:-methyltransferase (TPMT) genetic polymorphism has a significant clinical impact on the toxicity of thiopurine drugs, which are used in the treatment of leukemia and as immunosuppressants. To date, 10 mutant alleles are known that are associated with intermediate or low TPMT activity. To facilitate rapid screening of clinically relevant TPMT mutations, we developed a strategy of high-throughput genotyping by applying denaturing HPLC (DHPLC). To test the specificity and efficiency of the DHPLC method, 98 DNA samples from a selected population of patients receiving thiopurine therapy or with previous thiopurine withdrawal were analyzed for the most frequent mutant TPMT alleles, *2 and *3A, which contain key mutations in exons 5, 7, and 10 to identify clearly different elution profiles. All fragments were examined by direct sequencing. Additionally, to test the sensitivity of DHPLC analysis, genotyping for the *2 and *3A alleles of all 98 DNA samples was performed by PCR-based methods (PCR-restriction fragment polymorphism analysis and allele-specific PCR). The presence of mutations discriminating for alleles *2, *3A, *3C, and *3D, as well as various silent and intron mutations, were correctly predicted by DHPLC in 100% of the samples as confirmed by direct sequencing. Comparison with PCR-based methods for alleles *2 and *3 produced an agreement of 100% with no false-negative signals. DHPLC offers a highly sensitive, rapid, and efficient method for genotyping of the relevant TPMT mutations, discriminating at least for alleles *2 and *3, in clinical and laboratory practice. Additionally, DHPLC allows a simultaneous screening for novel genetic variability in the TPMT gene.

Frequency of thiopurine S‐methyltransferase genetic variation in Thai children with acute leukemia

Background Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation (inactivation) of mercaptopurine, azathioprine, and thioguanine, and exhibits genetic variation. About 11% of Caucasians have intermediate TPMT activity because of heterozygosity, and about 1 in 300 inherits TPMT deficiency as an autosomal codominant trait. If patients who have intermediate or deficient TPMT activity receive the standard dose of thiopurine medications, they can accumulate excessive thiopurine nucleotides in hematopoietic tissue, which could lead to severe and possibly fatal myelosuppression. There is very little information about TPMT genetic variation among Asian populations. We investigated the frequency of TPMT genetic variation among Thai children with acute leukemia. Procedure Fresh whole blood was obtained from 75 Thai children with acute leukemia at the time of remission. Genomic DNA was isolated from total peripheral white blood cells. We performed polymerase chain reaction (PCR) to detect 3 types of variant of the human TPMT gene. Results Among 75 patients, the frequency of heterozygotes for the TPMT gene among Thai children with acute leukemia was ∼11%. However, the TPMT*3C was the only variant TPMT allele found among Thai children. This is different from the North American Caucasian populations, in which TPMT*3A is the predominant variant allele, and TPMT*3C is rare (∼5% of variant alleles). Conclusions There is no difference in the frequency of this genetic variation between Asian and North American Caucasian populations. Determination of the TPMT genotype by PCR method before antileukemic therapy is practical and may have clinical relevance. This knowledge could be applied towards organ transplant recipients who require these medications for immunosuppression. Med. Pediatr. Oncol. 35:410–414, 2000. © 2000 Wiley-Liss, Inc.

Frequency of thiopurine S-methyltransferase genetic variation in Thai children with acute leukemia.

Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation (inactivation) of mercaptopurine, azathioprine, and thioguanine, and exhibits genetic variation. About 11% of Caucasians have intermediate TPMT activity because of heterozygosity, and about 1 in 300 inherits TPMT deficiency as an autosomal codominant trait. If patients who have intermediate or deficient TPMT activity receive the standard dose of thiopurine medications, they can accumulate excessive thiopurine nucleotides in hematopoietic tissue, which could lead to severe and possibly fatal myelosuppression. There is very little information about TPMT genetic variation among Asian populations. We investigated the frequency of TPMT genetic variation among Thai children with acute leukemia. Fresh whole blood was obtained from 75 Thai children with acute leukemia at the time of remission. Genomic DNA was isolated from total peripheral white blood cells. We performed polymerase chain reaction (PCR) to detect 3 types of variant of the human TPMT gene. Among 75 patients, the frequency of heterozygotes for the TPMT gene among Thai children with acute leukemia was approximately 11%. However, the TPMT*3C was the only variant TPMT allele found among Thai children. This is different from the North American Caucasian populations, in which TPMT*3A is the predominant variant allele, and TPMT*3C is rare (approximately 5% of variant alleles). There is no difference in the frequency of this genetic variation between Asian and North American Caucasian populations. Determination of the TPMT genotype by PCR method before antileukemic therapy is practical and may have clinical relevance. This knowledge could be applied towards organ transplant recipients who require these medications for immunosuppression.

Isolation of a human thiopurine S-methyltransferase (TPMT) complementary DNA with a single nucleotide transition A719G (TPMT*3C) and its association with loss of TPMT protein and catalytic activity in humans.

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes the S-methylation of mercaptopurine, azathioprine, thioguanine and most of their nucleotide metabolites. TPMT exhibits genetic polymorphism, with about 10% of individuals having intermediate TPMT activity because of heterozygosity at the TPMT locus and about 1 in 300 inheriting TPMT deficiency as an autosomal recessive trait. Although several mutant alleles have now been associated with inheritance of TPMT deficiency in humans, the expression of only TPMT*2 and TPMT*3A has been established by isolation and characterization of complementary DNA (cDNA) from individuals with low TPMT activity. Radiochemical assay, Western blot analysis, polymerase chain reaction (PCR) genotyping, and cDNA sequencing were used to analyze TPMT activity and protein levels in erythrocytes and to determine TPMT genotype. We established expression of another common mutant allele, TPMT*3C (containing only the A719G mutation), by sequence analysis of cDNA isolated from an individual with a heterozygous TPMT phenotype (7 units/ml packed erythrocytes). The TPMT*3C allele was also confirmed in an unrelated individual by sequencing TPMT coding exons after PCR amplification of genomic DNA. Moreover, Western blot analysis of erythrocytes obtained from five heterozygous individuals with the TPMT*3C allele (i.e., TPMT*1/TPMT*3C) exhibited about 50% less immunodetectable TPMT protein compared with homozygous wild-type individuals, and a TPMT-deficient individual with a TPMT*3A/TPMT*3C genotype had no immunodetectable TPMT protein. These data establish that the TPMT*3C allele is expressed in humans and is associated with lower immunodetectable TPMT protein and catalytic activity.

Molecular diagnosis of thiopurine S-methyltransferase deficiency: genetic basis for azathioprine and mercaptopurine intolerance.

Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation (that is, inactivation) of mercaptopurine, azathioprine, and thioguanine and exhibits genetic polymorphism. About 10% of patients have intermediate TPMT activity because of heterozygosity, and about 1 in 300 inherit TPMT deficiency as an autosomal recessive trait. If they receive standard doses of thiopurine medications (for example, 75 mg/m2 body surface area per day), TPMT-deficient patients accumulate excessive thioguanine nucleotides in hematopoietic tissues, which leads to severe and possibly fatal myelosuppression. To elucidate the genetic basis and develop molecular methods for the diagnosis of TPMT deficiency and heterozygosity. Diagnostic test evaluation. Research hospital. The TPMT phenotype was determined in 282 unrelated white persons, and TPMT genotype was determined in all persons who had intermediate TPMT activity (heterozygotes) and a randomly selected, equal number of persons who had high activity. In addition, genotype was determined in 6 TPMT-deficient patients. Polymerase chain reaction (PCR) assays were developed to detect the G238C transversion in TPMT*2 and the G460A and A719G transitions in TPMT*3 alleles. Radiochemical assay was used to measure TPMT activity. Mutations of TPMT were identified in genomic DNA, and the concordance of TPMT genotype and phenotype was determined. 21 patients who had a heterozygous phenotype were identified (7.4% of sample [95% CI, 4.7% to 11.2%]). TPMT*3A was the most prevalent mutant allele (18 of 21 mutant alleles in heterozygotes; 85%); TPMT*2 and TPMT*3C were more rare (about 5% each). All 6 patients who had TPMT deficiency had two mutant alleles, 20 of 21 patients (95% [CI, 76% to 99.9%]) who had intermediate TPMT activity had one mutant allele, and 21 of 21 patients (100% [CI, 83% to 100%]) who had high activity had no known TPMT mutation. Detection of TPMT mutations in genomic DNA by PCR coincided perfectly with genotypes detected by complementary DNA sequencing. The major inactivating mutations at the human TPMT locus have been identified and can be reliably detected by PCR-based methods, which show an excellent concordance between genotype and phenotype. The detection of TPMT mutations provides a molecular diagnostic method for prospectively identifying TPMT-deficient and heterozygous patients.