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

Abstract

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.

Thiopurine S-methyltransferase pharmacogenetics: insights, challenges and future directions

“…A total of 21 TPMT genetic polymorphisms have been identified which are, or may be associated with decreased levels of TPMT enzyme activity and/or thiopurine drug-induced toxicity . Eighteen of those polymorphisms (*2, *3A, *3B, *3C, *5-*14 and *16-*19) involve nonsynonymous coding single-nucleotide polymorphisms (cSNPs) (Krynetski et al, 1995;Szumlanski et al, 1996;Otterness et al, 1997;Lindqvist et al, 2004;Schaeffeler et al, 2004;Hamdan-Khalil et al, 2005), that is, alterations in single DNA nucleotides that alter the encoded amino acid. The *14 SNP disrupts the translation initiation codon (Lindqvist et al, 2004) and prevents translation of the enzyme protein , while *4 and *15 involve alterations in canonical mRNA splice site sequences (Otterness et al, 1998;Lindqvist et al, 2004) and *3D contains a premature stop codon (Otterness et al, 1997).…”

“…As might be anticipated, many techniques have been used to genotype TPMT, beginning with RFLP assays and later extending to allele-specific amplification, direct sequencing, SSCP, DHPLC and -more recently -use of a variety of high throughput platforms (Yates et al, 1997;Spire-Vayron de la Moureyre et al, 1998;Schaeffeler et al, 2003Schaeffeler et al, , 2004. However, beyond all of the usual issues associated with genotyping, TPMT pharmacogenetics serves to highlight -in a very practical sense -a limitation common to all of these genotyping methods, their inability to determine haplotype directly.…”

Pharmacogenetics and the concept of individualized medicine

“…24 Previous studies also have shown that patients with homozygous mutant TPMT alleles exhibit very low enzyme activity and develop a severe hematopoietic toxicity after treatment with standard doses of thiopurines. [25][26][27] Similarly, the response rate of 5-fluorouracil (5-FU) based treatment of advanced colorectal cancer is significantly linked to 677 C-T polymorphism in the methylenetetrahydrofolate reductase gene. 28 …”

Clinical significance of polymorphism of thiopurine methyltransferase gene in children with acute lymphoblastic leukemia during programmed therapy

“…В настоящее время известно более 20 полиморфных вариантов гена, при этом наибольшее клиническое значение у детей с гемобластозами придается следу-ющим генетическим аллелям: ТРМТ*3А, ТРМТ*3С, ТРМТ*2 [8][9][10][11]. Отмечается выраженная вариабель-ность встречаемости дефицита ТРМТ в зависимости от этнической принадлежности, в европейской попу-ляции частота выявления гетерозиготного носитель-ства аллелей составляет около 10 %, гомозиготного -около 1 % [8,10,12].…”