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 similar to11%) in the Bulgarians. The most common variant allele was TPMT-3A, as in other white populations

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, 37degreesC) 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 mum, 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