Enhanced proteasomal degradation of mutant human thiopurine S-methyltransferase (TPMT) in mammalian cells: mechanism for TPMT protein deficiency inherited by TPMT*2, TPMT*3A, TPMT*3B or TPMT*3C

Inheritance of the TPMT*2, TPMT*3A and TPMT*3C mutant alleles is associated with deficiency of thiopurine S-methyltransferase (TPMT) activity in humans. However, unlike TPMT*2 and TPMT*3A, the catalytically active protein coded by TPMT*3C does not undergo enhanced proteolysis when heterologously expressed in yeast, making it unclear why this common mutant allele should be associated with inheritance of TPMT-deficiency. To further elucidate the mechanism for TPMT deficiency associated with these alleles, we characterized TPMT proteolysis following heterologous expression of wild-type and mutant proteins in mammalian cells. When expressed in COS-1 cells, proteins encoded by TPMT*2, TPMT*3A, and TPMT*3C cDNAs had significantly reduced steady-state levels and shorter degradation half-lives compared with the wild-type protein. Similarly, in rabbit reticulocyte lysate (RRL), these mutant TPMT proteins were degraded significantly faster than the wild-type protein. Thus, enhanced proteolysis of TPMT*3C protein in mammalian cells is in contrast to its stability in yeast, but consistent with TPMT-deficiency in humans. Proteolysis was ATP-dependent and sensitive to proteasomal inhibitors MG115, MG132 and lactacystin, but not to calpain inhibitor II. We conclude that all of these mutant TPMT proteins undergo enhanced proteolysis in mammalian cells, through an ATP-dependent proteasomal pathway, leading to low or undetectable levels of TPMT protein in humans who inherit these mutant alleles.     

Functional characterization of 23 allelic variants of thiopurine S-methyltransferase gene (TPMT*2 - *24)

OBJECTIVE: Thiopurine S-methyltransferase (TPMT) is an enzyme responsible for the detoxification of the widely used thiopurine drugs. TPMT is genetically polymorphic and is associated with large interindividual variations in thiopurine drug toxicity and therapeutic efficacy. In this study, we performed an in-vitro analysis of TPMT variant alleles, namely, TPMT*2, *3A, *3B, *3C, *5, *6, *7, *8, *9, *10, *11, *12, *13, *14, *16, *17, *18, *19, *20, *21, *22, *23, and *24. METHODS: The wild-type TPMT proteins, TPMT.1 and 23 variants were heterologously expressed in COS-7 cells, and the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of 6-thioguanine S-methylation were determined. RESULTS: The expression levels of TPMT.2, TPMT.3A, TPMT.5, TPMT.12, TPMT.14, and TPMT.22 were considerably lower than that of TPMT.1 (P<0.005), and that of TPMT.18 was slightly reduced (P<0.05). The kinetic parameters of TPMT.3A, TPMT.3B, TPMT.5, TPMT.14, TPMT.18, TPMT.21, and TPMT.22 could not be accurately established because of no activity in 6-thioguanine S-methylation. The Vmax/Km values of TPMT.2, TPMT.7, TPMT.17, and TPMT.24 were displayed less than 10% of the wild-type. CONCLUSION: This functional analysis with respect to TPMT variants could provide useful information for individualization of thiopurine drugs therapy.     

Allelic variants of the thiopurine methyltransferase (TPMT) gene in the Colombian population

Thiopurine methyltransferase (TPMT) catalyzes the inactivation of thiopurine drugs (mercaptopurine, thioguanine and azathioprine) used to treat acute lymphoblastic leukemia, autoimmune diseases and recipients of transplanted organs. No endogenous substrates for this enzyme are known. The TPMT polymorphism is a major determinant of individual differences in the toxicity or therapeutic efficacy of these drugs. The molecular basis of this polymorphism has been established in Caucasians, Africans, African-Americans and Asians, but not yet in the heterogeneous Latin American groups, including the Colombian population. 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 140 Colombian volunteers of Mestizo origin, using allele-specific PCR and PCR-RFLP assays. The *3A allele was found in 10 samples and the *2 allele in one, all heterozygotes; neither homozygous mutant genotypes nor the *3B and *3C alleles were detected. In agreement with these results, 92.1% and 7.9% of the Colombian population correspond to the phenotypes high and intermediate methylators, respectively. These results show that the frequency of mutations and the allelic distribution of the TPMT gene in the Colombian population are similar to the genetic profile found among US and European Caucasian populations, where the *3A allele is prevalent and the *2 allele is currently present.     

中国新疆维吾尔族硫嘌呤甲基转移酶基因突变研究

目的 研究硫嘌呤甲基转移酶(thiopurine S-methyltransferase，TPMT)在新疆维吾尔族中的基因突变频率。方法 用等位基因特异性的PCR方法和限制性片断长度多态性的方法检测4种常见的导致酶活性降低的突变类型：TPMT*2、TPMT*3A、TPMT*3B和TPMT*3C。结果 在160名维吾尔族中发现了1例TPMT*3A(A719G／G460A)杂合子、5例TPMT*3C(A719G)杂合子，TPMT*3A和TPMT*3C的等位基因频率分别是0．3％和1．6％。结论 维吾尔族总的TPMT突变等位基因频率(1．9％)同中国其他民族相近；TPMT*3C是维吾尔族最主要的突变类型。     

河南地区汉族人群药物代谢酶CYP2C19，NAT2和TPMT基因多态性分析(英文)

目的：了解细胞色素P450(cytochromes P450,CYP)2C19,N-乙酰基转移酶2(arylamine N- acetyltransferase 2,NAT2)和硫嘌呤甲基转移酶(thiopurine S-methyltransferase,TPMT)基因常见的遗传多态性在河南地区汉族人群中的分布及其频率。方法：应用聚合酶链反应-限制性片段长度多态性分析(PCR-RFLP)对210名河南地区汉族人群的CYP2C19突变基因(*2和*3)、NAT2突变基因(*6和*7)和TPMT突变基因(*3A,*3B和*3C)进行检测。用聚合酶链反应-等位基因特异性扩增(PCR-ASA)对NAT2突变基因(*5)和TPMT突变基因(*2)进行检测。结果：CYP2C19*2和*3等位基因分布频率分别为34．76%和6．4%,同时携带2个等位突变基因的慢基因型频率占14．8%。NAT2*4(wt),*5(341C),*6(590A)和*7(857A)等位基因分布频率分别为59．1%,4．1%,26．4%和9．5%,慢基因型分布频率占19．5%。TPMT*3C等位基因分布频率为1．2%,未发现TPMT*2,TPMT*3A或TPMT*3B。结论：CYP2C19,NAT2和TPMT基因常见的遗传多态性在汉族人群中的分布及其频率与白人存在明显差异,这将有助于我国汉族人群临床药动学研究和给药剂量的确定。     

Genotyping of thiopurine methyltransferase using pyrosequencing

Thiopurine methyltransferase (TPMT) metabolizes thiopurine drugs which are used in the treatment of leukemia and some autoimmune diseases. Previously, 11 mutant alleles of TPMT gene (TPMT*1S, *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, and *8) have been reported. These mutant alleles may cause life-threatening toxicity in patients exposed to thiopurine drugs, 6-mercaptopurine and azathioprine. We have developed a rapid and accurate protocol for TPMT genotype determination using Pyrosequencing(TM) technology in 96 Japanese subjects. Five fragments of the TPMT gene (exon 4, 5, 7, 8, 10) were amplified by PCR, and the 10 single-nucleotide polymorphisms (SNPs) for TPMT*1S, *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, and *8 were sequenced. The results of this pyrosequencing method corresponded exactly with those of the DNA sequencing method using BigDye terminator chemistry. We have demonstrated that typing of 10 SNPs can be performed within 30 min. Pyrosequencing has a wide application in the large-scale identification of individual TPMT genotypes.     

The frequency and distribution of thiopurine methyltransferase alleles in Caucasian and Asian populations

Thiopurine methyltransferase metabolizes 6-mercaptopurine, thioguanine and azathioprine, thereby regulating cytotoxicity and clinical response to these thiopurine drugs. In healthy Caucasian populations, 89-94% of individuals have high thiopurine methyltransferase activity, 6-11% intermediate and 0.3% low, resulting from genetic polymorphism. Four variant thiopurine methyltransferase alleles were detected in over 80% of individuals with low or intermediate thiopurine methyltransferase activity. The wild-type allele is defined as TPMT*1 and the mutant alleles are TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3B (A719G). The frequency of these alleles in different ethnic groups is not well defined. In this study, DNA from 199 British Caucasian, 99 British South West Asian and 192 Chinese individuals was analysed for the presence of these variant alleles using polymerase chain reaction-restriction fragment length polymorphism and allele-specific polymerase chain reaction based assays. The frequency of individuals with a variant thiopurine methyltransferase genotype was: Caucasians 10.1% (20/199), South West Asians 2.0% (2/99) and Chinese 4.7% (9/192). Two TPMT*2 heterozygotes were identified in the Caucasian population, but this allele was not found in the two Asian populations. TPMT*3A was the only mutant allele found in the South West Asians (two heterozygotes). This was also the most common mutant allele in the Caucasians (16 heterozygotes and one homozygote) but was not found in the Chinese. All mutant alleles identified in the Chinese population were TPMT*3C (nine heterozygotes). This allele was found at a low frequency in the Caucasians (one heterozygote). This suggests that A719G is the oldest mutation, with G460A being acquired later to form the TPMT*3A allele in the Caucasian and South West Asian populations. TPMT*2 appears to be a more recent allele, which has only been detected in Caucasians to date. These ethnic differences may be important in the clinical use of thiopurine drugs.     

Thiopurine S-methyltransferase genetic polymorphism in the Thai population

AIMS: To determine the incidence of the thiopurine S-methyltransferase (TPMT) genetic polymorphism in the Thai population. METHODS: Genomic DNAs were isolated from peripheral blood leucocytes of 200 healthy Thais. The frequencies of five allelic variants of the TPMT gene, TPMT*2, *3A, *3B, *3C and *6 were determined using allele specific polymerase chain reaction (PCR) or PCR-Restriction fragment length polymorphism technique. RESULTS: Of the 200 Thai subjects participating in this study, 181 subjects (90.5%) were homozygous for TPMT*1, 18 subjects (9.0%) were heterozygous for TPMT*1/*3C. Only one subject (0.5%) was homozygous for TPMT*3C. The frequency of TPMT*3C mutant allele was 0.050. CONCLUSIONS: Although the TPMT*3C is the most prevalent mutant allele in Asian populations, the frequency of this defective allele is significantly higher in Thais than has been reported in other Asian populations.     

Explaining TPMT genotype/phenotype discrepancy by haplotyping of TPMT*3A and identification of a novel sequence variant, TPMT*23

Thiopurine methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism of thiopurine drugs. Owing to polymorphisms in the TPMT gene (TPMT*2-*22), the enzyme activity varies interindividually. Patients with reduced TPMT activity may develop adverse reactions when treated with standard doses of thiopurines. This work focuses on a TPMT genotype/phenotype discrepancy found in a patient during routine testing. The patient displayed very low TPMT enzyme activity and she was genotyped by pyrosequencing as being heterozygous for the 460G>A and 719A>G polymorphisms (TPMT*3A). Complete sequencing in combination with haplotyping of the TPMT gene revealed a novel sequence variant, 500C>G, on one allele and TPMT*3A on the other allele, giving rise to the novel genotype TPMT*3A/*23. When investigating the patient's relatives, they too had the TPMT*3A/*23 genotype in combination with low enzyme activity. We conclude that this novel variant allele affects enzyme activity, as the individuals carrying it had almost undetectable TPMT activity.     

Phenotyping and genotyping study of thiopurine S-methyltransferase in healthy Chinese children: a comparison of Han and Yao ethnic groups

AIMS: Ethnicity is an important variable influencing drug response. Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurine drugs. Previous population studies have identified ethnic variations in both phenotype and genotype of TPMT, but limited information is available within Chinese population that comprises at least 56 ethnic groups. The current study was conducted to compare both phenotype and genotype of TPMT in healthy Han and Yao Chinese children. METHODS: TPMT activity was measured in healthy Chinese children by a HPLC assay (n = 213, 87 Han Chinese and 126 Yao Chinese). Allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) were used to determine the frequency of TPMT mutant alleles (TPMT*2, TPMT*3 A, TPMT*3B and TPMT*3C) in these children. RESULTS: There was no significant difference in the mean TPMT activity between Han and Yao Chinese children. A unimodal distribution of TPMT activity in Chinese children was found and the mean TPMT activity was 13.32 +/- 3.49 U ml(-1) RBC. TPMT activity was not found to differ with gender, but tended to increase with age in Yao Chinese children. TPMT*2, TPMT*3B and TPMT*3A were not detected, and only one TPMT*3C heterozygote (Han child) was identified in 213 Chinese children. Erythrocyte TPMT activity of this TPMT*3C heterozygote was 12.36 U ml(-1) RBC. The frequency of the known mutant TPMT alleles was 0.2%[1/426] in Chinese children. CONCLUSION: The frequency distribution of RBC TPMT activity was unimodal. The frequency of the known mutant TPMT alleles in Chinese Children is low and TPMT*3C appears to be the most prevalent among the tested mutant TPMT alleles in this population.     

Post-translational stabilization of thiopurine S-methyltransferase by S-adenosyl-L-methionine reveals regulation of TPMT*1 and *3C allozymes

Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) plays a pivotal role in thiopurine treatment outcomes. However, little has been known about its intracellular regulation. Here, we describe the effect of fluctuations in physiological levels of S-adenosyl-L-methionine (SAM) and related metabolites on TPMT activity levels in cell lines and erythrocytes from healthy donors. We determined higher TPMT activity in wild-type TPMT*1/*1 individuals with high SAM concentrations (n=96) compared to the low SAM level group (n=19; P<0.001). These findings confirm the results of our in vitro studies, which demonstrated that the restriction of L-methionine (Met) in cell growth media reversibly decreased TPMT activity and protein levels. Selective inhibition of distinct components of Met metabolism was used to demonstrate that SAM is implicitly responsible for direct post-translational TPMT stabilization. The greatest effect of SAM-mediated TPMT stabilization was observed in the case of wild-type TPMT*1 and variant *3C allozymes. In addition to TPMT genotyping, SAM may serve as an important biochemical marker in individualization of thiopurine therapy.     

Genotype and allele frequencies of TPMT,NAT2, GST,SULT1A1 and MDR-1 in the Egyptian population

AIMS: The goal of this study was to determine the frequencies of important allelic variants in the TPMT, NAT2, GST, SULT1A1 and MDR-1 genes in the Egyptian population and compare them with the frequencies in other ethnic populations. METHODS: Genotyping was carried out in a total of 200 unrelated Egyptian subjects. TPMT*2 was detected using an allele-specific polymerase chain reaction (PCR) assay. TPMT*3C and NAT2 variants (*5,*6 and *7) were detected using an allele-specific real-time PCR assay. Detection of GSTM1 and GSTT1 null alleles was performed simultaneously using a multiplex PCR assay. Finally, a PCR-restriction fragment length polymorphism assay was applied for the determination of TPMT*3A (*3B), SULT1A1*2 and MDR-1 (3435T) variants. RESULTS: Genotyping of TPMT revealed frequencies of 0.003 and 0.013 for TPMT*3A and TPMT*3C, respectively. No TPMT*2 or *3B was detected in the analysed samples. The frequencies of specific NAT2 alleles were 0.215, 0.497, 0.260 and 0.028 for *4 (wild-type), *5 (341C), *6 (590A) and *7 (857A), respectively. GSTM1 and GSTT1 null alleles were detected in 55.5% and 29.5% of the subjects, respectively. SULT1A1*2 was detected at a frequency of 0.135. Finally, the frequencies of the wild-type allele (3435C) and the 3435T variant in the MDR-1 gene were found to be 0.6 and 0.4, respectively. CONCLUSIONS: We found that Egyptians resemble other Caucasians with regard to allelic frequencies of the tested variants of NAT2, GST and MDR-1. By contrast, this Egyptian population more closely resemble Africans with respect to the TPMT*3C allele, and shows a distinctly different frequency with regard to the SULT1A1*2 variant. The predominance of the slow acetylator genotype in the present study (60.50%) could not confirm a previously reported higher frequency of the slow acetylator phenotype in Egyptians (92.00%), indicating the possibility of the presence of other mutations not detectable as T341C, G590A and G857A. The purpose of our future studies is to investigate for new polymorphisms, which could be relatively unique to the Egyptian population.     

中国哈萨克族人硫嘌呤甲基转移酶活性分布和基因多态性

目的 研究硫嘌呤甲基转移酶(TPMT)在中国哈萨克族的活性分布和4 种常见TPMT基因突变等位基因频率。方法 用高效液相色谱法测定TPMT 活性;用等位基因特异性聚合酶链反应检测TPMT*2;用限制性片段长度多态性 检测TPMT*3A、TPMT*3B和TPMT*3C的等位基因频率。结果 哈萨克族T PMT活性呈正态分布,活性的均值为(12．27±3．42)U·mL-1 Rbc,其中发现6 例TPMT*3C杂合子和2例TPMT*3A杂合子,总TPMT基因突变频率是1．2％。 结论 中国哈萨克族TPMT活性呈正态分布,总TPMT基因突变频率同汉族相 比差异无显著性。     

Identification and functional analysis of two rare allelic variants of the thiopurine S-methyltransferase gene, TPMT*16 and TPMT*19

Human thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. TPMT is genetically polymorphic and is associated with large interindividual variations in thiopurine drug toxicity and therapeutic efficacy. During routine genotyping of patients with Crohn's disease, one novel missense mutation, 365A>C (TPMT*19, Lys(122)Thr), and a recently described missense mutation, 488G>A (TPMT*16, Arg(163)His), were identified in a Caucasian and a Moroccan patient, respectively. Using a heterologous yeast expression system, kinetic parameters (K(m) and V(max)) of the two variants with respect to 6-thioguanine S-methylation were determined and compared with those obtained with the wild-type enzyme. The Lys(122)Thr exchange did not significantly decrease the intrinsic clearance value (V(max)/K(m)) of the variant enzyme. In contrast, the Arg(163)His substitution significantly decreased the intrinsic clearance value by three-fold. The Arg(163) is located in a highly conserved region of the human TPMT protein and, as such, the Arg(163)His substitution is expected to result in a marked reduction of enzyme activity, as confirmed by the in vitro data. Phenotyping by measurement of red blood cell TPMT activity indicated that the patient heterozygous for the Lys(122)Thr mutation had normal TPMT activity, whereas the patient heterozygous for the Arg(163)His mutation was an intermediate methylator, which demonstrated a positive correlation between TPMT phenotyping and the in vitro data. The identification of a novel non-functional allele of the TPMT gene improves our knowledge of the genetic basis of interindividual variability in TPMT activity. These data further enhance the efficiency of genotyping methods to predict patients at risk of an inadequate response to thiopurine therapy.     

Thiopurine S-methyltransferase pharmacogenetics: genotype to phenotype correlation in the Slovenian population

The toxicity of thiopurine drugs has been correlated to the activity of thiopurine S-methyltransferase (TPMT), whose interindividual variation is a consequence of genetic polymorphisms. We have herein investigated the relevance of some genetic markers for the prediction of thiopurine-related toxicities and to determine the genotype to phenotype correlation in the Slovenian population. The most prevalent mutant allele in the Slovenian population is TPMT*3A (4.1%), followed by TPMT*3C (0.5) and TPMT*3B (0.3), while the TPMT*2 allele was not found in any of the examined samples. TPMT enzyme activity distribution in the subgroup sample was bimodal and as such correlated with genetic data. Using a cutoff value of 9.82 pmol/10(7) RBC per h, the genetic data correctly predicted TPMT enzyme activity in 91.6% of the examined individuals. Pharmacogenetic TPMT analyses have therefore proved to have significant clinical implications for prediction of individuals' responses to treatment with thiopurine drugs in order to avoid possible life-threatening therapy-related toxicities.     

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.     

Allelotype frequency of the thiopurine methyltransferase (TPMT) gene in Japanese

Polymorphisms at three loci in the thiopurine methyltransferase (TPMT) gene are known to be responsible for azathioprine and 6-mercaptopurine (6MP) toxicity. Among them, only TPMT*3C variant allele with A719G mutation was found in 15/522 (2.9%; 17/1044 alleles; 1.6%) Japanese individuals including two homozygotes. The allele frequency was different from that in Caucasians, and investigation of TPMT polymorphisms with consideration of ethnic differences before administration of azathioprine or 6MP may provide clinically useful information.     

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.     

基于连接酶反应检测硫唑嘌呤基因多态性及应用

目的：建立并探讨基于连接酶反应检测硫嘌呤甲基转移酶（thiopufine S．methyltransferase,TPMT）基因分型分布的新方法。方法：建立多重PCR／连接酶检测方法检测TPMT基因多态性,并对50份健康志愿者样本进行检测。结果：本方法可靠、迅速,自动化程度高。研究未发现导致TPMT酶活性降低的基因型＊2,＊3A,＊3B和＊4,TPMT ＊3C仅发现2例杂合子。结论：本文建立的基因分型技术可为TPMT基因型检测提供可靠的方法。     

Phenotypic and genotypic analysis of thiopurine s-methyltransferase polymorphism in the bulgarian population

Genetic polymorphism of TPMT activity is an important factor responsible for large individual differences in thiopurine toxicity and therapeutic efficacy. The aim of this study was to determine the distribution of TPMT activity as well as the types and frequencies of mutant alleles in a Bulgarian population sample. TPMT activity was measured in 313 Bulgarians, using an established HPLC procedure. All individuals with TPMT activity less than 12.0 nmol/(mL Ery.h) (n = 76) were additionally genotyped using a color multiplex hybridization assay. The samples were tested for TPMT*2, *3A, *3B, *3C, *3D, *4, and *6 mutant alleles. TPMT activities varied from 1.1 to 24.0 nmol/(mL Ery.h) [mean 14.2 +/- 3.2 nmol/(mL Ery.h)]: 92.3% of the individuals investigated had high TPMT activity [>10 nmol/(mL Ery. h)], whereas 7.4% were intermediate [2.8-10 nmol/(mL Ery.h)], and 0.3% were low metabolizers [< 2.8 nmol/(mL Ery.h)]. A significant gender-related difference in TPMT activity (P = 0.02) was observed with 6.2% higher values in men than in women. There was no significant correlation between age and enzyme activity (r = 0.06, P = 0.27). Genotype analysis revealed three mutant TPMT alleles: 2, 3A, and 3C. The frequency of these alleles among the TPMT-deficient individuals was 2.17%, 30.4%, and 2.17%, respectively. These data show a similar distribution of TPMT activity among the Bulgarian population investigated as in most other white populations with the frequency of intermediate metabolizers being somewhat lower (7.4% versus approximately 11%) in the Bulgarians. The most common variant allele was TPMT-3A, as in other white populations.