Determination of thiopurine methyltransferase phenotype in isolated human erythrocytes using a new simple nonradioactive HPLC method

Genetic polymorphism of the S-methylation pathway catalyzed by thiopurine methyltransferase (TPMT) is responsible for variation in the metabolism, toxicity, and therapeutic efficacy of thiopurine drugs. This paper describe a new simple, nonradioactive HPLC method for determination of TPMT activity in isolated erythrocytes (Ery), based on the conversion of 6-mercaptopurine (pH 7.5, 37 degrees C) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-l-methionine as methyl donor. The incubation step was stopped by a mixture of trichloroacetic acid/acetonitrile containing the internal standard 4-aminoacetophenone. 6-MMP was quantified by absorbance at 290 nm after chromatographic separation on a Zorbax SB-Phenyl column (5 microm, 4.6 x 250 mm) using mobile phases (flow rate 1.1 mL/min) consisting of acetonitrile, phosphate buffer pH 3.0, triethylamine, and dithiothreitol. The assay was linear up to 50 nmol/(mL Ery. h), and the detection limit was 0.3 nmol/(mL Ery. h). The extraction efficiency of 6-MMP was 95-103% (n = 3), and its analytic recovery ranged between 98.3% and 101.8% (n = 12). The within-day imprecision using pooled human erythrocytes (n = 12) was 4.4% at a TPMT activity of 14.3 nmol/(mL Ery.h) and 4.9% at 6.5 nmol/(mL Ery.h). The between-day imprecision (n = 12) was 6.8% and 7.5% nmol/(mL Ery.h), respectively. A very good agreement was found between TPMT activity determined with this method (y) and a widely used radiochemical procedure (x) (r = 0.94; n = 130; y = 0.502 + 0.946x; P < 0.05). Genotype analysis of all individuals with TPMT activity under 12.5 nmol/(mL Ery.h) revealed a genotype/phenotype concordance of 86%. The new HPLC method for determination of TPMT activity in Ery is a simple, rapid, and reliable nonradioactive procedure that can be successfully used for both research and routine clinical analysis.     

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.     

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 methyltransferase phenotypes and genotypes in Brazilians

The polymorphism of thiopurine methyltransferase (TPMT) was studied in 306 healthy Brazilians who were classed, on the basis of self-declared colour and ancestry, as Euro-derived (n = 81), Afro-derived (n = 18) or having interethnic admixture (n = 204). TPMT activity (range 0.17-25.93 U) displayed a trimodal distribution of high (> 11.3 U; 9% of individuals), intermediate (5-11.3 U; 9.8%) and low (0.17 U; 0.3%) phenotypes. The occurrence of the TPMT mutations 238G>C, 460G>A and 719A>G was investigated in all individuals with low or intermediate phenotype, and in 43 with high-activity phenotype. None and two mutant alleles were associated with high- or low-activity phenotypes, respectively, whereas one mutant allele was detected in 26 of the 30 intermediate phenotype individuals. The allele frequencies of TPMT*2, TPMT*3A and TPMT*3C did not differ between individuals classed as Euro-derived (0.76%, 2.03% and 2.54%, respectively) or having interethnic admixture (0.60%, 1.81% and 1.81%, respectively). Furthermore, within each of these groups, the frequencies of TPMT*3A and TPMT*3C were not significantly different.     

Ethnic differences in thiopurine methyltransferase pharmacogenetics: evidence for allele specificity in Caucasian and Kenyan individuals

Thiopurine methyltransferase (TPMT) degrades 6-mercaptopurine, azathioprine and 6-thioguanine which are commonly used in the treatment of autoimmune diseases, leukaemia and organ transplantation. TPMT activity is polymorphic as a result of gene mutations. Heterozygous individuals have an increased risk of haematological toxicity after thiopurine medication, while homozygous mutant individuals suffer life threatening complications. Previous population studies have identified ethnic variations in both phenotype and genotype, but limited information is available within African populations. This study determined the frequency of common TPMT variant alleles in 101 Kenyan individuals and 199 Caucasians. The frequency of mutant alleles was similar between the Caucasian (10.1%) and Kenyan (10.9%) populations. However, all mutant alleles in the Kenyan population were TPMT*3C compared with 4.8% in Caucasians. In contrast TPMT*3A was the most common mutant allele in the Caucasian individuals. This study confirms ethnic differences in the predominant mutant TPMT allele and the findings will be useful for the development of polymerase chain reaction-based strategies to prevent toxicity with thiopurine medications.     

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.     

Genotype–phenotype correlation for thiopurine S-methyltransferase in healthy Italian subjects

OBJECTIVE: The aim of the present study was to estimate the concordance rate between erythrocyte thiopurine methyltransferase (TPMT) activity and genotype at the TPMT locus in an Italian population sample. METHODS: The TPMT phenotype and genotype were determined in an unrelated population of 103 Italian healthy blood donors. Erythrocyte TPMT activity was measured with a radiochemical assay using 12.5 microM S-adenosyl-L-(methyl-14C)-methionine and 4 mM 6-mercaptopurine. The genotyping assay was based on restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) methods. RESULTS: All subjects had detectable TPMT activity. The activity of TPMT varied 2.8-fold between the 5th and 95th percentile. This variation was neither age (P = 0.63) nor gender (P = 0.44) regulated and the frequency distribution of TPMT activity is compatible with a polymorphic distribution. The presence of the four most common defective alleles, i.e. TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C, was examined through the entire phenotyped population. Ninety-two subjects did not carry any of the tested mutations. Eleven individuals were heterozygous for one of the mutant alleles and had a TPMT activity lower than 30 pmol/min/mg. Eight subjects were TPMT*1/TPMT*3A, two TPMT*1/TPMT*3C and one was TPMT*1/TPMT*2. The TPMT*3B allele was not detected in the samples analysed. CONCLUSION: There was a concordance of 97% between genotype and phenotype. All the heterozygotes had an intermediate phenotype. However, the wide variation range in TPMT activity detected in the wild-type homozygotes indicates that other genetic or epigenetic factors influence the TPMT phenotype.     

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.     

Phenotype and genotype for thiopurine methyltransferase activity in the French Caucasian population: impact of age

Objective Thiopurine drugs are commonly used in pediatric patients for the treatment of acute leukemia, organ transplantation and inflammatory diseases. They are catabolized by the cytosolic thiopurine methyltransferase (TPMT), which is subject to a genetic polymorphism. In children, enzyme activities are immature at birth and developmental patterns vary widely from one enzyme to another. The present study was undertaken to evaluate erythrocyte TPMT activity and the correlation between genotype and phenotype in different age groups from birth to adolescence and adulthood.Methods The study included 304 healthy adult blood donors, 147 children and 18 neonates (cord bloods). TPMT activity was measured by liquid chromatography, and genotype was determined using a polymerase chain reaction reverse dot-blot analysis identifying the predominant TPMT mutant alleles (TPMT*3A, TPMT*3B, TPMT*3C, TPMT*2).Results There was no significant difference in TPMT activity between cord bloods (n=18) and children (n=147) (17.48±4.04 versus 18.62±4.14 respectively, P=0.424). However, TPMT was significantly lower in children than in adults (19.34±4.09) (P=0.033). In the whole population, there were 91.9% homozygous wild type, 7.9% heterozygous mutants and 0.2% homozygous mutants. The frequency of mutant alleles was 3.0% for TPMT*3A, 0.7% for TPMT*2 and 0.4% for TPMT*3C.Conclusion No impact of child development on TPMT activity could be evidenced, suggesting that TPMT activity is already mature at birth. The difference between children and adults was low with reduced clinical impact expected. When individual TPMT activity was compared with genotype, there was an overlapping region where subjects (4.5%, 12 adults, 9 children) were either homozygous wild type or heterozygous, with a TPMT activity below the antimode value. This result highlighted the importance of measuring TPMT activity to detect all patients at risk of thiopurine toxicity.     

Genetic determinants of the thiopurine methyltransferase intermediate activity phenotype in British Asians and Caucasians

OBJECTIVE: Polymorphisms in the TPMT gene open reading frame (ORF) are associated with reduced TPMT activity. Variable number tandem repeats (VNTR*3 to VNTR*9) in the promoter region of the gene consisting of combinations of Type A, B and C repeat units, may modulate TPMT activity. Here we present the allele frequencies of genetic modifiers of TPMT activity in a British Asian population, as well as the concordance between intermediate TPMT activity and ORF and VNTR genotypes in a predominantly Caucasian population. METHODS: VNTR type and ORF mutations were determined in two selected TPMT activity ranges, intermediate activity (4-8 U, 108 patients), normal (12-15 U, 53 patients) and in 85 British Asians. RESULTS: In British Asians, TPMT*3C was the prevalent mutant allele (four heterozygotes). One patient was heterozygous for TPMT*3A. Overall VNTR frequencies did not differ from Caucasians. Three new VNTR alleles were designated VNTR*6c, VNTR*6d, and VNTR*7c. Forty-one percent of patients with intermediate activity were heterozygous for a TPMT ORF mutation (3A, 2B, 1C). Marked linkage disequilibrium was noted between VNTR*6b - TPMT*3A (D' = 1), VNTR*4b - TPMT*3C (D' = 0.67) and VNTR*6a - TPMT*1 (D' = 1) alleles. As a result, significant differences (P < 0.05) in the distribution of Type A, B or the total number of repeats summed for both alleles, were found between the ORF heterozygous intermediate activity group and the wild-type intermediate or normal activity groups. No significant difference was found between the two wild-type groups. CONCLUSION: Our results suggest that TPMT gene VNTRs do not significantly modulate enzyme activity.     

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.     

Thiopurine S-methyltransferase phenotype-genotype correlation in hemodialyzed patients

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme, catalyzing S-methylation of thiopurine drugs. TPMT exhibits autosomal codominant polymorphism. Patients carrying a variant genotype have low TPMT activity, and produce elevated levels of 6-thioguanine nucleotides (6-TGN) in their red blood cells (RBC). 6-TGN accumulation may result in azathioprine (AZA)-induced bone marrow myelosuppression in the course of treatment with the drug in a standard dosage regimen in patients following renal transplantation. In the current study, TPMT activity (phenotype) and genotype were determined in dialyzed patients, qualified for renal transplantation. TPMT activity was measured in RBC after dialysis by HPLC method. Patients were genotyped for TPMT *2, *3A and *3C variant alleles using PCR-RFLP and allele-specific PCR methods. TPMT activity ranged between 12.2 and 45.5 nmol 6-mMP/g Hb/h (median value 30.6). A significant correlation between TPMTphenotype and genotype was noted: the heterozygous patients (11.5%) demonstrated significantly lower mean TPMT activity as compared to the wild homozygotes (17 +/- 3.6 vs. 32.4 +/- 4.8 nmol 6-mMP/g Hb/h, p < 0.0003). No overlap in TPMT activity values between the group of heterozygous (range 12.2-20.6) and wild-type homozygous patients (range 22.7-45.5) was noted. TPMT activity, established after hemodialysis and TPMT genotyping results seem to be convergent in dialyzed patients, so both methods can be used for the identification of patients with lower TPMT activity. Such tests could be helpful in AZA dose individualization, and thus in reducing the risk of myelosuppression during AZA therapy following renal transplantation.     

Thiopurine methyltransferase genotype and phenotype status in Japanese patients with systemic lupus erythematosus

We investigated the genotypic status of thiopurine methyltransferase (TPMT) polymorphism to evaluate the possible risk of the toxicity of azathioprine (AZA) in 68 patients with systemic lupus erythematosus (SLE). The allele frequency of TPMT mutation in the SLE group (2.9%) was higher than that in 174 Japanese healthy volunteers (1.1%), although it did not reach statistically significant difference (p=0.23). The mean value of TPMT activities in 51 subjects with TPMT*1/*1 was 40% higher than that of 4 subjects with TPMT*1/*3C in SLE group (18.1+/-6.1 nmol/h/ml packed red blood cells (pRBC) versus 13.2+/-3.2 nmol/h/ml pRBC; p=0.11). Two out of 4 SLE patients with TPMT*1/*3C had been treated with AZA, and one patient showed a leucopenia. The TPMT genotyping before AZA treatment is recommended for Japanese SLE patient group to avoid the AZA-induced adverse events, although detection of the patient with low TPMT activity by genotyping is still imperfect.     

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.     

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。     

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。     

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.     

Thiopurine<Emphasis Type="Italic"> S</Emphasis>-methyltransferase polymorphisms: efficient screening method for patients considering taking thiopurine drugs

Objective More than 11% of the Caucasian population are heterozygous or homozygous carriers of thiopurine S-methyltransferase (TPMT) mutants and are at risk for toxic side effects when treated with thiopurine drugs. Therefore, screening for TPMT polymorphisms in a patient prior to prescribing these agents is recommended. The goal of this study was to determine a cut-off concentration of the TPMT activity assay beyond which genotyping of the TPMT gene should be performed.Methods The TPMT activity of 240 unrelated Caucasian subjects was measured using high-performance liquid chromatography. Genotyping for the most frequent allelic variants, TPMT*2, *3A, *3B, *3C and *7 was performed by LightCycler technology and sequencing.Results The inter-individual TPMT activity showed a range from 23 nmol MTG/g*Hb*h^–1 to 97 nmol MTG/g*Hb*h^–1 with a median of 56 nmol MTG/g*Hb*h^–1. Using a cut-off concentration of 45.5 nmol MTG/g*Hb*h^–1, a test sensitivity of 100% and a specificity of 89% were reached for heterozygous carriers of a TPMT mutation. We identified 1 carrier of TPMT*2, 14 carriers of TPMT*3A and 3 carriers of TPMT*3C, resulting in a TPMT heterozygosity prevalence of 7.5%.Conclusions This study defines the cut-off value for the TPMT phenotyping assay at 45.5 nmol/g*Hb*h^–1, beyond which additional genotyping elucidates the individual risk for drug therapy. Using this cut-off concentration, the number of genotyping assays could be reduced by about 60%.     

Frequencies of thiopurine S-methyltransferase mutant alleles (TPMT*2, *3A, *3B and *3C) in 151 healthy Japanese subjects and the inheritance of TPMT*3C in the family of a propositus

AIMS: To determine the frequencies of four thiopurine S-methyltransferase (TPMT) mutant alleles, TPMT*2, *3A, *3B and *3C in a normal Japanese population. METHODS: Genotypes were determined in 151 Japanese subjects and in six family members of a propositus using polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific PCR assays. RESULTS: Only one TPMT*3C heterozygote was identified (gene frequency 0.3%). TPMT*2, *3A and *3B were not detected. In addition, TPMT*3C was found to have been inherited from the mother and passed on to the son of the propositus. CONCLUSIONS: TPMT*3C appears to be most prevalent among the known mutant allele of TPMT in a Japanese population which may have some relevance for the treatment of Japanese patients with thiopurine drugs.     

Analysis of the CYP2D6 gene mutations and their consequences for enzyme function in a West African population

The data on differences in the metabolic handling of the CYP2D6 probe drugs sparteine and debrisoquine, and the relationship between phenotype and genotype and gene frequencies for several mutant CYP2D6 alleles in African populations are limited and sometimes controversial. Therefore, in a West African population (Ghana), we investigated (i) the phenotype for sparteine debrisoquine by phenotyping 201 individuals with both drugs and (iii) the genotype for CYP2D6 (n = 326) and debrisoquine (n = 201) oxidation, (ii) the coregulatory control of sparteine and alleles *3 and *4 in 133 individuals and for the alleles *1, *2, *3, *4, *5, *6, *7, *8, *9, *10, *14, *16, *17, *2b, *2xN, *2bxN in 193 individuals. Of the 326 individuals phenotyped with sparteine, eight had a metabolic ratio (MR)sp > 20 corresponding to a poor metabolizer frequency of 2.5% [95% (confidence interval) CI = 1.06-4.77]. The prevalence of the poor metabolizer phenotype for debrisoquine oxidation was 3% (95% CI = 1.1-6.39) with six of the 201 individuals having a MR greater than 12.6. The distribution of the MR of sparteine was trimodal whereas MR of debrisoquine was unimodally distributed with a pronounced kurtosis. In individuals phenotyped with both drugs, there was a significant correlation between the MRs (r(s) = 0.63, P < 0.001). The CYP2D6 alleles *1, *2 and *17 were the most common functional alleles occurring with frequencies of 43.7, 10.6 and 27.7%, respectively. The three other observed functional alleles *2xN, *10 and *20 had much lower frequencies (1.6%, 3.1% and 0.3%, respectively). Of the eight non-functional alleles, only *4 (6.3%) and *5 (6.0%) could be found. The allele *5 occurred with the same frequency as in Caucasian populations (4.1%) but the *4 allele had a much lower frequency (Caucasians 19.5%). One individual with *1/*1 was a poor metabolizer for sparteine and debrisoquine indicating the existence of as yet unknown non-functional alleles in this West African population. Although the prevalence of poor metabolizers and the number of heterozygotes for non-functional alleles was much lower in Ghanaians, the median MRsp of 0.7 was significantly higher in this population compared with a median MRsp of 0.4 in Caucasians, indicating a lower metabolic clearance for CYP2D6 substrates in the West Africans. The lower metabolic activity in Ghanaians could not be explained solely by the high frequency of the *17 allele, which is associated with an impairment of CYP2D6 enzyme function. In addition, a higher median MRsp of 0.5 corresponding to metabolic clearance of 346 ml/min was observed among extensive metabolizers with the genotype *1/*1. Thus, compared with the median of MRsp = 0.28 (CLmet 573 ml/min) in Caucasians homozygous for *1, the metabolic clearance of sparteine was 40% lower on average in respective Ghanaians.