Thiopurine S-methyltransferase pharmacogenetics: chaperone protein association and allozyme degradation

Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs such as 6-mercaptopurine. A common genetic polymorphism for TPMT is associated with large individual variations in thiopurine drug toxicity and therapeutic efficacy. TPMT*3A, the most common variant allele in Caucasians, has two alterations in amino acid sequence, resulting in striking decreases in TPMT protein levels. This phenomenon results, in part, from rapid degradation through a ubiquitin-proteasome-mediated process. We set out to test the hypothesis that chaperone proteins might be involved in targeting TPMT for degradation. As a first step, hsp90, hsp70 and the cochaperone hop were immunoprecipitated from a rabbit reticulocyte lysate (RRL) that included radioactively labelled *3A and wild-type TPMT. TPMT*3A was much more highly associated with all three chaperones than was the wild-type enzyme. The RRL was also used to confirm the accelerated degradation of *3A compared to wild-type TPMT. Treatment of RRL with the hsp90 inhibitor geldanamycin resulted in enhanced association of hsp90 with wild-type TPMT, an observation that correlated with accelerated ubiquitin-dependent degradation of wild-type TPMT. Geldanamycin treatment of COS-1 cells transfected with FLAG-tagged wild-type also resulted in a time and geldanamycin concentration-dependent decrease in TPMT activity and protein, which was compatible with results obtained in the RRL. These observations indicate that TPMT is a client protein for hsp90 and suggest that chaperone proteins, especially hsp90, are involved in targeting both TPMT*3A and, in the presence of geldanamycin, the wild-type allozyme for degradation. Therefore, chaperone proteins play an important mechanistic role in this clinically significant example of pharmacogenetic variation in drug metabolism.     

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.     

Thiopurine S-methyltransferase pharmacogenetics: Functional characterization of a novel rapidly degraded variant allozyme

A novel human thiopurine S-methyltransferase (TPMT) variant allele, (319 T > G, 107Tyr > Asp, *27), was identified in a Thai renal transplantation recipient with reduced erythrocyte TPMT activity. The TPMT*27 variant allozyme showed a striking decrease in both immunoreactive protein level and enzyme activity after transient expression in a mammalian cell line. We set out to explore the mechanism(s) responsible for decreased expression of this novel variant of an important drug-metabolizing enzyme. We observed accelerated degradation of TPMT*27 protein in a rabbit reticulocyte lysate. TPMT*27 degradation was slowed by proteasome inhibition and involved chaperone proteins—similar to observations with regard to the degradation of the common TPMT*3A variant allozyme. TPMT*27 aggresome formation was also observed in transfected mammalian cells after proteasome inhibition. Inhibition of autophagy also decreased TPMT*27 degradation. Finally, structural analysis and molecular dynamics simulation indicated that TPMT*27 was less stable than was the wild type TPMT allozyme. In summary, TPMT*27 serves to illustrate the potential importance of protein degradation - both proteasome and autophagy-mediated degradation - for the pharmacogenetic effects of nonsynonymous SNPs.     

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.     

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.     

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.     

Characterisation of novel defective thiopurine S-methyltransferase allelic variants

Human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) is a key enzyme in the detoxification of thiopurine drugs widely used in the treatment of various diseases, such as inflammatory bowel diseases, acute lymphoblastic leukaemia and rheumatic diseases. The TPMT gene is genetically polymorphic and the inverse relationship between TPMT activity and the risk of developing severe hematopoietic toxicity is well known. In this study, the entire coding sequence of TPMT, together with its 5'-flanking promoter region, was analysed in patients with an intermediate phenotype for thiopurine drug methylation. Four polymorphisms were identified, two previously described, c.356A>C (p.Lys(119)Thr, TPMT*9) and c.205C>G (p.Leu(69)Val, TPMT*21), and two novel missense mutations, c.537G>T (p.Gln(179)His, TPMT*24) and c.634T>C (p.Cys(212)Arg, TPMT*25). Structural investigations, using molecular modeling, were undertaken in an attempt to explain the potential impact of the amino acid substitutions on the structure and activity of the variant proteins. Additionally, in order to determine kinetic parameters (K(m) and V(max)) of 6-thioguanine (6-TG) methylation, the four variants were expressed in a recombinant yeast expression system. Assays were performed by HPLC and the results were compared with those of wild-type TPMT. The p.Leu(69)Val and the p.Cys(212)Arg substitutions encode recombinant enzymes with a significantly decreased intrinsic clearance compared to that of the wild-type protein, and, consequently, characterise non-functional alleles of TPMT. The p.Lys(119)Thr and the p.Gln(179)His substitutions do not affect significantly the catalytic activity of the corresponding variant proteins, which prevents to unambiguously describe these latter alleles as defective TPMT variants.     

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.     

The impact of thiopurine s-methyltransferase polymorphism on azathioprine-induced myelotoxicity in renal transplant recipients

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 as a result of genetic polymorphism. Patients with intermediate or deficient TMPT 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 low enzyme activity. The purpose of this study was to explore the association between these polymorphisms and the occurrence of azathioprine adverse effects in 112 renal transplant recipients undergoing triple immunosuppressive therapy including azathioprine, cyclosporine, and prednisone. TPMT genetic polymorphism was determined using PCR-RFLP and allele-specific PCR methods. Azathioprine dose, leukocyte, erythrocyte, and platelet counts, graft rejection episodes, as well as cyclosporine levels were analyzed throughout the first year after organ transplantation. We found the frequency of leukopenia episodes (WBC < 4.0 x 10(9)/L) significantly higher in heterozygous patients (53.8%) compared with those with TPMT wild-type genotype (23.5%). One patient, who was a compound homozygote (3A/*3C), experienced severe azathioprine-related myelotoxicity each time after receiving the standard drug dose. Our results suggest that polymorphisms in TPMT gene may be responsible for approximately 12.5% of all leukopenia episodes in renal transplant recipients treated with azathioprine. Genotyping for the major TPMT variant alleles may be a valuable tool in preventing AZA toxicity and optimization of immunosuppressive therapy.     

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: autophagy as a mechanism for variant allozyme degradation

OBJECTIVE: Thiopurine S-methyltransferase (TPMT)*3A is degraded much more rapidly than is the 'wild-type' enzyme through a ubiquitin-proteasome-dependent process. It also forms aggresomes, suggesting a possible dynamic balance between degradation and aggregation. We set out to identify genes encoding proteins participating in these processes. METHODS: Green fluorescent protein tagged TPMT*3A was expressed in a Saccharomyces cerevisiae gene deletion library, and flow cytometry was used to screen for cells with high fluorescence intensity, indicating the loss of a gene essential for TPMT*3A degradation. RESULTS: Twenty-four yeast genes were identified in functional categories that included ubiquitin-dependent protein degradation, vesicle trafficking, and vacuolar degradation. The presence of genes encoding proteins involved in vesicular transport and vacuolar degradation suggested a possible role in TPMT*3A degradation for autophagy--a process not previously identified as a pharmacogenomic mechanism. In support of that hypothesis, TPMT*3A aggregates increased dramatically in mutants for vacuolar protease and autophagy-related genes. Furthermore, TPMT*3A expression in human cells induced autophagy, and small interfering RNA-mediated knockdown of ATG7, an autophagy-related human protein, enhanced TPMT*3A aggregation but not that of TPMT*3C or wild-type TPMT, indicating that autophagy contributes to TPMT*3A degradation in mammalian cells. We also demonstrated that UBE2G2, the human homologue of the E2 ubiquitin-conjugating enzyme identified during the yeast genetic screen, was involved in TPMT*3A degradation in human cells. CONCLUSION: These results indicate that autophagy should be considered among mechanisms responsible for the effects of pharmacogenetically significant polymorphisms that alter encoded amino acids.     

Thiopurine S-methyltransferase phenotype-genotype correlation in children with acute lymphoblastic leukemia

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 as a result of genetic polymorphism. Patients with intermediate or deficient TPMT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. The aim of this study was to determine the TPMT genotype and phenotype (activity) and investigate the correlation between TPMT genotype and enzyme activity in 43 Polish children receiving 6-MP during maintenance therapy in course of acute lymphoblastic leukemia (ALL), in 16 children with ALL at diagnosis and 39 healthy controls. TPMT activity was measured in RBC by HPLC method. Patients were genotyped for TPMT *2, *3A and *3C variant allelesusing PCR-RFLP and allele-specific PCR methods. In the group of children with ALL during maintenance therapy, median TPMT activity (29.3 nmol 6-mMP g(-1) Hb h(-1)) was significantly higher compared to the group of children with ALL at diagnosis (20.6 nmol 6-mMP g(-1) Hb h(-1), p = 0.0028), as well as to the control group (22.8 nmol 6-mMP g(-1) Hb h(-1), p = 0.0002). Percentages of individuals heterozygous for TPMT variant allele in respective groups were: 9.3, 6.2 and 15.5% (p > 0.05). In all the study groups heterozygous patients manifested a significantly lower TPMT activity as compared to the wild type homozygotes (16.7 +/- 2.1 vs. 31.2 +/- 6.8 nmol 6-mMP g(-1) Hb h(-1), p = 0.002, in children during maintenance therapy, 11.9 +/- 2.7 vs. 24.6 +/- 9.5, p = 0.0003, in the combined group of children with ALL at diagnosis and controls). The results present that commencement of the thiopurine therapy caused an increase in the TPMT activity in RBCs by approximately 20%. All patients heterozygous for the TPMT variant allele revealed decreased TPMT activity compared to TPMT wild-type patients. Since decreased TPMT activity is associated with higher risk for toxicity after receiving standard doses of thiopurine drugs, pretreatment determination of TPMT status, with phenotypic or genetic assay, should be performed routinely, also in Poland.     

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.     

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。     

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.     

Human kidney thiopurine methyltransferase. Photoaffinity labeling with S-adenosyl-L-methionine.

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of heterocyclic and aromatic sulfhydryl compounds such as the thiopurine drug 6-mercaptopurine (6-MP). TPMT activity in human tissue is regulated by a common genetic polymorphism, and "pharmacogenetic" variation in TPMT activity is an important factor in individual differences in thiopurine drug metabolism, toxicity and therapeutic efficacy. Human renal tissue contains two isozymes of TPMT, Peak I and Peak II, that can be separated by ion exchange chromatography. Our experiments were performed to determine whether S-adenosyl-L-methionine (Ado-Met), the methyl donor for the TPMT reaction, could be used as a photoaffinity ligand for these isozymes as one step in the study of the molecular basis for the TPMT genetic polymorphism. When [3H-methyl]Ado-Met and partially purified preparations of either isozyme of human kidney TPMT were exposed to ultraviolet light at 254 nm, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a 35 kDa protein was the predominant species that was radioactively labeled. The same 35 kDa protein was photoaffinity labeled with [14C-carboxyl]Ado-Met, demonstrating that labeling involved covalent binding of Ado-Met rather than methylation of the protein. TPMT enzymatic activity co-eluted with the 35 kDa protein during sequential DEAE ion exchange, gel filtration and hydroxylapatite chromatography. Inhibitors of TPMT enzymatic activity including S-adenosyl-L-homocysteine, sinefungin, 6-methylmercaptopurine and 3,4-dimethoxy-5-hydroxybenzoic acid inhibited photoaffinity labeling of the 35 kDa protein in preparations of both TPMT Peak I and Peak II isozymes in a concentration-dependent fashion, as did 6-MP, the methyl acceptor substrate for the TPMT reaction. All of these results were compatible with the conclusion that the 35 kDa protein was TPMT. Photoaffinity labeling of TPMT with [3H]Ado-Met should make it possible to purify the enzyme to homogeneity and to study amino acid sequences at or near its active site.     

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.     

Canine red blood cell thiopurine S-methyltransferase: companion animal pharmacogenetics

Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurine drugs. In humans, a common genetic polymorphism for TPMT is a major factor responsible for individual variation in the toxicity and therapeutic efficacy of these drugs. Dogs (Canis familiaris) are also treated with thiopurine drugs and, similar to humans, they display large individual variations in thiopurine toxicity and efficacy. We set out to determine whether dogs might also display genetically determined variation in TPMT activity. As a first step, we observed that canine red blood cell (RBC) TPMT activity in samples from 145 dogs varied over a nine-fold range. That variation was not associated with either the age or sex of the animal. Subsequently, we cloned the canine TPMT cDNA and gene. The canine cDNA encoded a protein that was 81.2% identical to the enzyme encoded by the most common TPMT allele in humans. A genotype-phenotype correlation analysis was performed by resequencing the canine gene using DNA samples from 39 animals selected for high, low or intermediate levels of RBC TPMT activity. We observed nine polymorphisms in these 39 DNA samples, including three insertion/deletion events and six single nucleotide polymorphisms (SNPs), one of which was a nonsynonymous cSNP (Arg97Gln). However, when the variant allozyme at codon 97 was expressed in COS-1 cells, it did not display significant differences in either basal levels of TPMT activity or in substrate kinetics compared with the wild-type allozyme. Six of the nine canine TPMT polymorphisms were associated with 67% of the variation in level of RBC TPMT activity in these 39 blood samples. When those six SNPs were assayed using DNA from all 145 animals studied, 40% of the phenotypic variance in the entire population sample could be explained by these polymorphisms. Therefore, inheritance is a major factor involved in the regulation of variation in RBC TPMT in the dog, just as it is in humans. These observations represent a step towards the application of pharmacogenetic and pharmacogenomic principles to companion animal drug therapy.     

Methylation pharmacogenetics: thiopurine methyltransferase as a model system.

1. Methyl conjugation is an important pathway in the biotransformation of many drugs and xenobiotic compounds. 'Pharmacogenetic' variation exists in the activities of many methyltransferase enzymes, and experiments with the drug-metabolizing enzyme thiopurine methyltransferase (TPMT) offer a model for one approach that has proven useful in the study of methyltransferase pharmacogenetics. 2. TPMT catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. This enzyme activity is present in the human red blood cell (RBC), and RBC TPMT activity is controlled by a common genetic polymorphism that regulates also the enzyme activity in all other human tissues that have been studied. 3. Subjects with inherited low levels of TPMT activity are at increased risk for thiopurine drug-induced myelotoxicity, while patients with high TPMT activities may be 'undertreated' with these drugs. 4. TPMT activity in tissue from selected strains of inbred mice also is regulated by a genetic polymorphism. These mice provide an animal model for use in the study of pharmacological or toxicological consequences of inherited differences in TPMT activity. 4. Other methyltransferase enzymes including thiol methyltransferase, catechol O-methyltransferase, and histamine N-methyltransferase also are present in the human RBC, are regulated by inheritance, and are responsible for individual variation in drug metabolism. Enhanced understanding of the pharmacogenetics of methylation may make it possible to understand and predict individual variation in the biotransformation, toxicity and therapeutic effect of compounds that undergo methyl conjugation.     

Human methylenetetrahydrofolate reductase pharmacogenomics: gene resequencing and functional genomics

5,10-Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in the folate metabolic pathway. Common genetic polymorphisms in the human MTHFR gene are associated with individual variation in the efficacy and toxicity of chemotherapeutic agents, such as methotrexate and 5-fluorouracil. However, the full range of polymorphisms and intragene haplotypes in the human MTHFR gene remains unclear. Furthermore, cellular mechanisms by which common, naturally occurring nonsynonymous coding single nucleotide polymorphisms (cSNPs) might alter the function of this enzyme have not been defined. The present study focused on the systematic identification and investigation of common polymorphisms and haplotypes in the MTHFR gene using a genotype-to-phenotype strategy, followed by functional genomic studies. Specifically, we resequenced exons, splice junctions and portions of the 5'-flanking region (5'-FR) of the human MTHFR gene using 240 DNA samples from four ethnic groups. A total of 65 polymorphisms were observed, 11 of which were nonsynonymous cSNPs. We then performed functional genomic studies with constructs for wild-type and 15 variant allozymes (some with multiple alterations in amino acid sequence) using a mammalian expression system. Activity for the variant allozymes ranged from 13% to 149% of wild-type activity. Levels of immunoreactive protein for the allozymes ranged from 31% to 120% of wild-type and were significantly correlated with enzyme activity (Rp=0.85, P<0.0001), suggesting that a major mechanism by which nonsynonymous cSNPs influence the function of this gene is by alteration in the quantity of protein. These observations represent steps towards an understanding of molecular genetic mechanisms responsible for variation in MTHFR function that may contribute to individual differences in drug efficacy and toxicity, as well as disease risk.