Printer Friendly

TPMT gene polymorphisms: on the doorstep of personalized medicine.

Since the initial human genome sequence has become available in 2001, sequence variability among individual genomes came into focus of the research in the field of human genetics. More than 99 per cent of the DNA sequence is identical among individuals. The remaining DNA is responsible for genetic diversity (1). Polymorphisms are common genetic variations in the human genome representing sequence variations that occur in >1 per cent of gene alleles in a given population. The most studied polymorphisms, SNPs (single nucleotide polymorphisms) were discovered thirty years ago. These are distributed over the whole genome. The number of SNPs is estimated to range from 0.5 to 1 SNP per 100 base pairs (bp). Besides SNPs, there are other important classes of polymorphisms, such as VNTRs (variable number of tandem repeats, polymorphic sequence containing 2050 copies of 6-100 bp repeats), STRs (short tandem repeats, a subclass of VNTR in which repeat unit consists of 2-7 bp) and copy number polymorphisms.

Despite extensive studies of polymorphisms, among a myriad of "promising" genetic markers, only a few have been shown to be valid ones. There is no doubt that the thiopurine S-methyltransferase (TPMT) gene polymorphism is one of them. Moreover it is already successfully applied at the bedside as a powerful pharmacogenetic marker.

TPMT gene polymorphisms are pharmacogenetic markers which enable the individualization of thiopurine drug therapy. Thiopurine drugs [6-mercaptopurine, (6-MP), thioguanine (TG) and azathioprine] are widely used in the treatment of many diseases, such as acute leukaemia, different types of inflammatory and autoimmune diseases and in transplantation medicine. Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme which catalyzes the S-methylation, and consequent partial inactivation of thiopurine drugs (2,3). Some patients treated with standard doses of thiopurine drugs accumulate high levels of TG nucleotides, usually leading to severe haematopoietic toxicity (4). Mostly it is a consequence of inherited TPMT deficiency. Approximately 90 per cent of individuals inherit both functional TPMT alleles resulting in high TPMT activity. Intermediate TPMT activity is seen in carriers of one nonfunctional, polymorphism affected, TPMT allele, representing 10 per cent of population. Low or undetectable TPMT activity is reported in 0.3 per cent individuals who inherit two nonfunctional TPMT alleles (5). Patients with low TPMT activity are at high risk of severe, eventually fatal, haematologic toxicity. Consequently, thiopurine drug dose reduction is necessary. Patients with intermediate TPMT activity also require dose reduction to avoid toxicity (6). Therefore, the level of TPMT enzyme activity is essential for balance of therapeutic and toxic effects of thiopurine drug dose.

TPMT gene exhibits significant genetic polymorphism. At present, a total of 25 TPMT genetic polymorhisms, mostly SNPs, have been identified (7). TPMT SNPs are, or may be associated with decreased levels of TPMT enzyme activity and thiopurine druginduced toxicity. Among these, the most common are: c.238G>C, c.460G>A and c.719A>G. There are several TPMT variant alleles comprising one or more SNPs. On the basis of population studies, three alleles account for more than 95 per cent of the clinically relevant TPMT variants: TPMT*3A, TPMT*3C and TPMT*2, with the last of them contributing to a lesser extent (8). Wild type has been designated as TPMT*1. TPMT*2 allele contains single c.238G>C polymorphism, TPMT*3A allele has two polymorphisms c.460G>A and c.719A>G, while TPMT*3C has only c.719A>G polymorphism.

It is important to emphasize that the distribution of clinically relevant alleles is population specific (9-12). TPMT*3A allele is the most common variant allele in Caucasians (frequency approximately 5%), while TPMT*3C is predominant in subjects with Asian or African ancestry (frequencies of 0.3-3% and 5.5-7.6% respectively).

Since inherited decrease of TPMT activity results in potentially life-threatening clinical consequences for patients treated with thiopurine drugs, a need for measurement of TPMT enzyme activity emerged. TPMT genotyping is highly sensitive and specific alternative to expensive TPMT enzyme activity determination. More than 98 per cent concordance exists between TPMT genotype and phenotype. Therefore, TPMT genotyping is a reliable method for guiding thiopurine therapy. It is of great importance that TPMT genotyping becomes a part of standard diagnostic protocols for diseases treated with thiopurine drugs. Prior to that, it was necessary to determine which TPMT polymorphisms should be tested in a certain population. In this issue, an application of a new technique (SNaPshot) for analysis of TPMT gene polymorphisms is presented (13). Kapoor and colleagues have developed a new approach (SNaPshot technique) for detection of three most common TPMT polymorphisms (c.238G>C, c.460G>A and c.719A>G) and applied it in Indian population.

Widely used methods for TPMT genotyping are ARMS (amplification refractory mutation system) and PCR-RFLP (polymerase chain reaction- restriction fragment length polymorphism) (14). Kapoor et al (13) chose the SNaPshot method, a non-time consuming, halfautomated method which enables testing of TPMT SNPs in a multiplex reaction. The method is based on a dideoxy single-base (ddNTP) extension of primers complementary to the sequences of three most common TPMT polymorphisms. The addition of one of four ddNTPs labeled with different fluorescent dyes at the position of the SNP is followed by electrophoresis and analysis of data.

The authors have designed a similar method in which cDNA is used as a template. Thiopurines are used in all phases of current therapy for childhood acute lymphoblastic leukaemia (ALL) (15, 16) and TPMT genotyping is necessary for appropriate adjustment of thiopurine drug doses. Since detection of several chimeric transcripts is a standard diagnostic procedure in childhood ALL, cDNA of each patient is available. Therefore, SNaPshot method for TPMT genotyping using cDNA template is especially interesting and useful.

The authors suggest that the main advantage of the SNaPshot approach is its up-scalability, as a high throughput platform for TPMT SNPs analysis with possible automation. An actual contribution of this specific diagnostic approach will be tested and confirmed in clinical laboratory practice, especially its cost-effectiveness. The new methodology was used to determine the frequency of three most common TPMT polymorphisms in the population of India. The overall frequency of TPMT polymorphisms was 4.9 per cent (13). The most common variant allele was TPMT*3C reaching the frequency of 4.1 per cent. Kham et al (17) have previously reported the frequency of 0.8 per cent for TPMT*3C among Indian migrant population in Singapure. However, this study is the first one reporting TPMT genotypes among resident (non-migrant) Indians (13). Since TPMT genotyping is recommended in clinical routine before administrating thiopurine therapy, the application of SNaPshot technique for analysis of TPMT gene polymorphisms represents an important progress towards introducing a rapid method of TPMT polymorphism detection in well equipped laboratories.

TPMT pharmacogenetics has been studied extensively because of its clinical significance. Many patients got benefited from the knowledge of TPMT genotype-phenotype correlation. However, further research is needed to elucidate the influence of TPMT polymorphism on drug metabolism (pharmacokinetics) and drug targets (pharmacodynamics) (18). Characterization of new TPMT polymorphisms and their effect on the level of enzyme activity will be a subject of future studies.

TPMT pharmacogenetics represents a great promise that personalized medicine, a dream of scientists and medical practitioners, will enter everyday medical practice in the near future.


SP was supported by grant 143 051 from the Ministry of Science and Technological Development, Serbia.


(1.) Kidd KK, Pakstis AJ, Speed WC, Kidd JR. Understanding human DNA sequence variation. J Hered 2004; 95 : 406-20.

(2.) Weinshilboum RM, Raymond FA, Pazmino PA. Human erythrocyte thiopurine methyltransferase: radiochemical microassay and biochemical properties. Clin Chim Acta 1978; 85 : 323-33.

(3.) Krynetski EY, Tai HL, Yates CR, Fessing MY, Loennechen T, Schuetz JD. Genetic polymorphism of thiopurine Smethyltransferase: clinical importance and molecular mechanisms. Pharmacogenetics 1996; 6 : 279-90.

(4.) Balis FM, Holcenberg JS, Poplack DG. General principles of chemotherapy. In: Pizzo AP, Poplack DG, editors. Principles and practice of pediatric ontology. 4th ed. Philadelphia: JB Lippincott Company; 2001. p. 210-45.

(5.) Weinshilboum RM, Sladek SL. Mercaptopurine pharmacogenetics: monogenic inheritance of erythrocyte thiopurine methyltransferase activity. Am J Hum Genet 1980; 32 : 651-62.

(6.) Dokmanovic L, Urosevic J, Janic D, Jovanovic N, Petrucev B, Tosic N, et al. Analysis of thiopurine S-methyltransferase polymorphism in the population of Serbia and Montenegro and mercaptopurine therapy tolerance in childhood acute lymphoblastic leukemia. Ther Drug Monit 2006; 28 : 800-6.

(7.) Garat A, Cauffiez C, Renault N, Lo-Guidice JM, Allorge D, Chevalier D, et al. Characterisation of novel defective thiopurine S-methyltransferase allelic variants. Biochem Pharmacol 2008; 76 : 404-15.

(8.) Szumlanski C, Otterness D, Her C, Lee D, Brandriff B, Kelsell D. Thiopurine methyltransferase pharmacogenetics: human gene cloning and characterization of a common polymorphism. DNA Cell Biol 1996; 15 : 17-30.

(9.) De la Moureyre SC, Debuysere H, Mustain B. Genotypic and phenotypic analysis of the polymorphic thiopurine Smethyltransferase gene (TPMT) in European population. Br J Pharmacol 1998; 125 : 879-87.

(10.) Hon YY, Fessing MY, Pui CH, Relling MV, Krynetski EY, Evans WE. Polymorphism of the thiopurine Smethyltransferase gene in African-Americans. Hum Mol Genet 1999; 8 : 371-6.

(11.) Collie-Duguid ES, Pritchard SC, Powrie RH, Sludden J, Collier DA, Li T, et al. The frequency and distribution of thiopurine methyltransferase alleles in Caucasian and Asian populations. Pharmacogenetics 1999; 9 : 37-42.

(12.) Schaffeler E, Fischer C, Brockmeier D, Wernet D, Moerike K, Eichelboum M, et al. Comprehensive analysis of thiopurine Smethyltransferase phenotype-genotype correlation in a large population of German-Caucasians and identification of novel TPMT variants. Pharmacogenetics 2004; 14 : 407-17.

(13.) Kapoor G, Maitra A, Somlata, Brahmachari V. Application of SNaPshot for analysis of thiopurine methyltransferase gene polymorphism, Indian J Med Res 2009; 129 : 500-5.

(14.) Yates CR, Krynetski EY, Loennechen T, Fessing MY, Tai HL, Pui CH, et al. Molecular diagnosis of thiopurine Smethyltransferase deficiency: genetic basis for azathioprine and mercaptopurine intolerance. Ann Intern Med 1997; 126 : 608-14.

(15.) Schrappe M, Camitta B, Pui CH, Eden T, Gaynon P, Gustafsson G, et al. Long-term results of large prospective trials in childhood acute lymphoblastic leukemia. Leukemia 2000; 14 : 2193-4.

(16.) Pui CH, Relling MV, Evans WE. Role of pharmacogenomics and pharmacodynamics in the treatment of acute lymphoblastic leukaemia. Best Pract Res Clin Haematol 2003; 15 : 741-56.

(17.) Kham SKY, Soh CK, Liu TC, Chan YH, Ariffin H, Tan PL, et al. Thiopurine S-methyltransferase activity in three major Asian populations: a population-based study in Singapore. Eur J Clin Pharmaco1 2008; 64 : 373-9.

(18.) Weinshilboum R, Wang L. Thiopurine S-methyltransferase pharmacogenetics: insights, challenges and future directions. Oncogene 2006; 25 : 1629-38.

Sonja Pavlovic

Institute of Molecular Genetics & Genetic Engineering

University of Belgrade

Belgrade, Serbia
COPYRIGHT 2009 Indian Council of Medical Research
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2009 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Commentary
Author:Pavlovic, Sonja
Publication:Indian Journal of Medical Research
Date:May 1, 2009
Previous Article:Salt never calls itself sweet: 'Jamaican saying'.
Next Article:Metabolic syndrome as a marker of risk in type 2 diabetes.

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters