Thiopurine Methyltransferase Deficiency Research Articles

Therapeutic drug monitoring of immunosuppressant drugs.

Individualizing a patient’s drug therapy to obtain the optimum balance between therapeutic efficacy and the occurrence of adverse events is the physician’s goal. However, achieving this goal is not always straight forward, being complicated by within and between patient variability in both pharmacokinetics and pharmacodynamics. In the early 1960s new analytical techniques became available allowing the measurement of the low drug concentrations seen in biological fluids during drug treatment. This offered the opportunity to reduce the pharmacokinetic component of variability by controlling drug therapy using concentrations in the body rather than by dose alone. This process became known as therapeutic drug monitoring (TDM) [1]. For a drug to be a suitable candidate for therapeutic drug monitoring it must satisfy the following criteria:- ✓ There should be a clear relationship between drug concentration and effect. ✓ The drug should have a narrow therapeutic index; that is, the difference in the concentrations exerting therapeutic benefit and those causing adverse events should be small. ✓ There should be considerable between-subject pharmacokinetic variability and, therefore, a poor relationship between dose and drug concentration/response. ✓ The pharmacological response of the drug should be difficult to assess or to distinguish from adverse events. The immunosuppressive drug cyclosporin satisfies all four of these criteria and, despite over 16 years of clinical use with therapeutic drug monitoring, there is still no firm consensus on the best way to use the drug. In addition, the number of available agents for use as immunosuppressants has more than doubled in recent years and the range of diseases in which these drugs are used has also widened [2]. The purpose of this review is to examine the current strategies in use for the therapeutic drug monitoring of immunosuppressant drugs [3] and to discuss some of the factors that impinge on the monitoring of these drugs. Azathioprine, steroids, anti-lymphocyte globulin, and OKT3 The combination of azathioprine and prednisolone was responsible for making clinical transplantation viable [4]. With the addition of anti-lymphocyte globulin [5] (ALG or ATG if human thymocytes instead of human lymphocytes are used to immunise the animal host) they formed the basis of immunosuppression in the early years of transplantation and these drugs are still in widespread use today. Monitoring the blood or plasma concentration of these drugs is not considered worthwhile as they all have relatively wide therapeutic indices. The three agents are generally given in fixed doses and are not subjected to therapeutic drug monitoring. However, a case can be made for the measurement of the activity of the enzyme thiopurine methyltransferase (TPMT) as an adjunct to azathioprine therapy [6]. Azathioprine is not directly immunosuppressive, since it must be metabolised first to 6-mercapto-purine, then by TPMT to 6-methyl-mercapto-purine and then on to the pharmacologically active 6-thioguanine nucleotides. The expression of the enzyme TPMT is inherited in an autosomal co-dominant fashion and consequently varies widely within the population [7] with 11% of the Caucasian population heterozygous and 0.3% homozygous with respect to TPMT deficiency [8]. Potentially fatal complications could be avoided if TPMT activity was monitored in erythrocytes [9]. The therapeutic drug monitoring of azathioprine in cancer chemotherapy is outside the scope of this article but has been reviewed recently in this journal by Lennard [10]. OKT3 (muromonab-CD3) is a mouse-monoclonal antibody directed against the CD3 complex on T cells [11]. When complexed with its antigen, the antibody prevents the initiation of signal transduction and blocks all T cell function [12]. In a pilot study using OKT3 serum concentrations as a guide to therapy in kidney transplant patients excellent results were reported for the prevention of early graft rejection [13]. Although there is a correlation between OKT3 concentration and T cell killing the relationship is complicated by the patients’ antibody response to murine-derived protein [14]. In another study using flow cytometry measurements to monitor OKT3 therapy the authors not only measured OKT3 concentration but also anti-OKT3 antibody concentration and the number of CD3+ cells (the therapeutic target of OKT3) [15]. Although the authors’ conclusions were positive about the use of flow cytometry for monitoring, their over all conclusions were that ‘this treatment cannot protect against acute cellular rejection due to the presence of a dimly positive CD3+ population’. In those centres using muromonab-CD3, TDM for OKT3 is not in widespread use.

Pharmacogenetics as a molecular basis for individualized drug therapy: the thiopurine S-methyltransferase paradigm.

Genetic polymorphism of drug metabolizing enzymes can be the major determinant of inter-individual differences in drug disposition and effects. In this mini-review, the evolution of pharmacogenetic studies, from the recognition of phenotypic polymorphisms to the discovery of genetic mutations responsible for these inherited traits, is illustrated by the genetic polymorphism of thiopurine S-methyltransferase (TPMT). TPMT, which exhibits autosomal co-dominant polymorphism, plays an important role in metabolism of the antileukemic and immunosuppressive medications, mercaptopurine, thioguanine, and azathioprine. The genetic polymorphism of TPMT activity in humans was first reported in 1980, and in the last five years the genetic basis for this polymorphism has been elucidated. Isolation and cloning of mutant alleles from humans with TPMT deficiency has identified the major mutant alleles, established the basis for loss of TPMT activity and permitted development of PCR-based genotyping assays to make a molecular diagnosis of TPMT-deficiency and heterozygosity. These studies illustrate the potential clinical benefits of elucidating the molecular basis of inherited differences in drug metabolism and disposition, and future automation of molecular diagnostics will make it feasible to more precisely select the optimal drug and dosage for individual patients.

Isolation of a human thiopurine S-methyltransferase (TPMT) complementary DNA with a single nucleotide transition A719G (TPMT*3C) and its association with loss of TPMT protein and catalytic activity in humans.

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes the S-methylation of mercaptopurine, azathioprine, thioguanine and most of their nucleotide metabolites. TPMT exhibits genetic polymorphism, with about 10% of individuals having intermediate TPMT activity because of heterozygosity at the TPMT locus and about 1 in 300 inheriting TPMT deficiency as an autosomal recessive trait. Although several mutant alleles have now been associated with inheritance of TPMT deficiency in humans, the expression of only TPMT*2 and TPMT*3A has been established by isolation and characterization of complementary DNA (cDNA) from individuals with low TPMT activity. Radiochemical assay, Western blot analysis, polymerase chain reaction (PCR) genotyping, and cDNA sequencing were used to analyze TPMT activity and protein levels in erythrocytes and to determine TPMT genotype. We established expression of another common mutant allele, TPMT*3C (containing only the A719G mutation), by sequence analysis of cDNA isolated from an individual with a heterozygous TPMT phenotype (7 units/ml packed erythrocytes). The TPMT*3C allele was also confirmed in an unrelated individual by sequencing TPMT coding exons after PCR amplification of genomic DNA. Moreover, Western blot analysis of erythrocytes obtained from five heterozygous individuals with the TPMT*3C allele (i.e., TPMT*1/TPMT*3C) exhibited about 50% less immunodetectable TPMT protein compared with homozygous wild-type individuals, and a TPMT-deficient individual with a TPMT*3A/TPMT*3C genotype had no immunodetectable TPMT protein. These data establish that the TPMT*3C allele is expressed in humans and is associated with lower immunodetectable TPMT protein and catalytic activity.

Pharmacokinetics, dose adjustments, and 6‐mercaptopurine/ methotrexate drug interactions in two patients with thiopurine methyltransferase deficiency

Two children with acute lymphoblastic leukaemia (ALL) were found to be thiopurine methyltransferase (TPMT)‐deficient by both genotype and phenotype. They were monitored with haematological parameters and red blood cell concentrations of 6‐thioguanine nucleotides (E‐6TGN) and methotrexate (E‐MTX, including MTX polyglutamates), in relation to the doses of 6‐mercaptopurine (6MP) and methotrexate (MTX), during their maintenance chemotherapy. Both patients developed severe pancytopenia at the standard protocol dose of 6MP. Even at 25% and 5%, respectively, of the protocol dose of 6MP, they achieved E‐6TGN values several‐fold above the population median, but without unacceptable bone‐marrow toxicity. Their high E‐6TGN values had only a minor influence on their E‐MTX values and their tolerance to oral MTX, but severe pancytopenia followed high‐dose MTX infusions. Due to the risk of fatal myelo‐suppression we recommend up‐front determination of TPMT activity in patients treated with 6MP or azathioprine.

Molecular Diagnosis of Thiopurine S-Methyltransferase Deficiency

Letters1 December 1997Molecular Diagnosis of Thiopurine S-Methyltransferase DeficiencyEugene Y. Krynetski, PhD and William E. Evans, PharmDEugene Y. Krynetski, PhDSt. Jude Children's Research Hospital; Memphis, TN 38101Search for more papers by this author and William E. Evans, PharmDSt. Jude Children's Research Hospital; Memphis, TN 38101Search for more papers by this authorAuthor, Article, and Disclosure Informationhttps://doi.org/10.7326/0003-4819-127-11-199712010-00027 SectionsAboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinkedInRedditEmail IN RESPONSE:We are pleased by Dr. Blank's interest in our paper and offer the following in response to points raised in his letter. We have previously shown that the G238C transversion alone is associated with a 100-fold reduction in TPM catalytic activity in a heterologous expression system [1] and that this is the only mutation in some patients with TPM deficiency [1]. Likewise, we have shown that the G460A and the A719G mutations are the only mutations in the TPM complementary DNA (cDNA) of other patients with TPM deficiency, and that the cDNA with only these mutations has more ...

Thiopurine methyltransferase deficiency in childhood lymphoblastic leukaemia: 6‐mercaptopurine dosage strategies

Daily 6-mercaptopurine (6MP) forms the backbone of continuing chemotherapy for childhood lymphoblastic leukaemia (ALL). A major metabolic route is catalysed by thiopurine methyltransferase (TPMT). TPMT deficiency occurs in 1 in 300 individuals and results in high concentrations of thioguanine nucleotides (TGNs), cytotoxic 6MP metabolites. A leukaemic child taking 6MP repeatedly developed profound pancytopenias. TPMT deficiency was confirmed. TGN formation was then studied on attenuated 6MP dosages. Four weekly oral doses of 75 mg/m2 6MP produced TGNs of 2348 pmol/8 × 108 red cells, nearly double the maximum TGNs recorded in ALL children with TPMT activity taking long term daily 75 mg/m2 6MP. Grossly elevated TGN concentrations were also produced at 10% standard 6MP dosage (7.5 mg/m2 daily), accompanied by unacceptable 6MP toxicity (neutropenia, diarrhoea, vomiting). The child was eventually stabilised on 10% alternate day therapy and after 15 weeks TGNs were 1670 pmol, just above the upper end of the TGN range for ALL children with TPMT activity. Med. Pediatr. Oncol. 29:252–255, 1997. © 1997 Wiley-Liss, Inc.

ACUTE LYMPHOBLASTIC LEUKEMIA

Advances in the molecular and immunologic characterization of leukemic cells have greatly aided the diagnosis and risk assignment of ALL, as well as the monitoring of bone marrow samples for minimal residual disease. Currently, 75% of childhood cases have biologically and therapeutically relevant genetic abnormalities. Although gene discoveries in ALL have not been directly translated into effective therapy, there is every reason to believe that this disease will eventually yield to molecular intervention. In the meantime, efforts are being made to enhance the efficacy of existing regimens while reducing their toxic side effects. We have learned, for example, the following: high-dose methotrexate is more effective than lower-dose methotrexate, especially for T-cell ALL; patients who need drastic adjustment of mercaptopurine dosage due to thiopurine S-methyltransferase deficiency can be prospectively identified; dexrazoxane (ICRF-187) could reduce anthracycline cardiotoxicity; granulocyte colony-stimulating factor can shorten hospital stays for febrile neutropenia after intensive remission induction therapy; and prolonged low-dose epipodophyllotoxin treatment may reduce the risk of therapy-induced acute myeloid leukemia without compromising treatment efficacy. The challenge now is to identify specific treatments for genetically defined subtypes of ALL.

Molecular diagnosis of thiopurine S-methyltransferase deficiency: genetic basis for azathioprine and mercaptopurine intolerance.

Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation (that is, inactivation) of mercaptopurine, azathioprine, and thioguanine and exhibits genetic polymorphism. About 10% of patients have intermediate TPMT activity because of heterozygosity, and about 1 in 300 inherit TPMT deficiency as an autosomal recessive trait. If they receive standard doses of thiopurine medications (for example, 75 mg/m2 body surface area per day), TPMT-deficient patients accumulate excessive thioguanine nucleotides in hematopoietic tissues, which leads to severe and possibly fatal myelosuppression. To elucidate the genetic basis and develop molecular methods for the diagnosis of TPMT deficiency and heterozygosity. Diagnostic test evaluation. Research hospital. The TPMT phenotype was determined in 282 unrelated white persons, and TPMT genotype was determined in all persons who had intermediate TPMT activity (heterozygotes) and a randomly selected, equal number of persons who had high activity. In addition, genotype was determined in 6 TPMT-deficient patients. Polymerase chain reaction (PCR) assays were developed to detect the G238C transversion in TPMT*2 and the G460A and A719G transitions in TPMT*3 alleles. Radiochemical assay was used to measure TPMT activity. Mutations of TPMT were identified in genomic DNA, and the concordance of TPMT genotype and phenotype was determined. 21 patients who had a heterozygous phenotype were identified (7.4% of sample [95% CI, 4.7% to 11.2%]). TPMT*3A was the most prevalent mutant allele (18 of 21 mutant alleles in heterozygotes; 85%); TPMT*2 and TPMT*3C were more rare (about 5% each). All 6 patients who had TPMT deficiency had two mutant alleles, 20 of 21 patients (95% [CI, 76% to 99.9%]) who had intermediate TPMT activity had one mutant allele, and 21 of 21 patients (100% [CI, 83% to 100%]) who had high activity had no known TPMT mutation. Detection of TPMT mutations in genomic DNA by PCR coincided perfectly with genotypes detected by complementary DNA sequencing. The major inactivating mutations at the human TPMT locus have been identified and can be reliably detected by PCR-based methods, which show an excellent concordance between genotype and phenotype. The detection of TPMT mutations provides a molecular diagnostic method for prospectively identifying TPMT-deficient and heterozygous patients.

Analysis of the human TPMT gene promoter

Background: Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs that are commonly used to treat a wide range of conditions. It is now well established that interpatient variation in sensitivity to these drugs is due to point mutations in the TPMT gene. The mutant alleles TPMT*2 (238G>C), TPMT*3A (460G>A, 719A>G), TPMT*3B (460G>A), and TPMT*3C (719A>G) account for 80–95% of TPMT deficiency observed in Caucasian populations. In this paper, we describe a novel, multiplex, allele-specific polymerase chain reaction (PCR) method that detects the 238G>C, 460G>A, and 719A>G mutations, allowing for the simultaneous identification of TPMT*2 and TPMT*3 alleles. The assay is internally controlled, robust, and does not require the use of restriction endonucleases. Therefore, the assay is not prone to erroneous readings due to incomplete restriction digestion, as documented for existing PCR restriction fragment length polymorphism (RFLP) assays of TPMT.

Azathioprine-induced myelosuppression due to thiopurinemethyltransferase deficiency in a patient with autoimmune hepatitis

Azathioprine can cause severe myelosuppression. The inherited activity of the enzyme thiopurine methyltransferase has been recently recognised as a major factor in the susceptibility to myelosuppression. Thiopurine methyltransferase deficiency occurs at a frequency of one in 300 and is associated with profound myelosuppression after a short course of azathioprine. Very low thiopurine methyltransferase activity represents the TPMT L/TPMT L genotype, and can be detected before therapy with azathioprine is started. We describe the first documented case of azathioprine-induced severe myelosuppression due to thiopurine methyltransferase deficiency in autoimmune liver disease. The azathioprine dose was low (1 mg/kg) and pancytopenia occurred after 56 days therapy. It would be advisable to measure thiopurine methyltransferase activity before patients with autoimmune hepatitis are exposed to azathioprine.

Monitoring of TPMT in Heart Transplant Recipients under Immunosuppressive Therapy with Azathioprine

Azathioprine (AZA) is routinely used in double and triple immunosuppressive therapy after cardiac transplantation. In some cases it causes severe myelosuppression. The interindividual differences in AZA toxicity is probably due to differences in the drug metabolism. Thiopurine methyltransferase (TPMT) is thought to be the most important enzyme in the catabolism of AZA. A deficiency in this enzyme will presumably increase the availability of 6-mercaptopurine for the anabolic pathway thereby leading to increased cytotoxicity. A 65-year-old male underwent heart transplantation at our institution with an uncomplicated course. Immunosuppression consisted of cyclosporine, prednisolone, and AZA. Several weeks after the administration of AZA, the patient developed severe leukopenia. TPMT activity was then measured in this patient and found to be below the detection limit. Subsequently the patient died from multiorgan failure due to septicaemia. As a result of this experience, we started to screen all patients for TPMT deficiency. In 58 healthy controls, the mean activity was found to be 11.8 nmol/h/ml of red blood cells (RBC) while in 13 patients on our waiting list, the mean activity was found to be 11.97 nmol/h/ml of RBC. In 15 patients after heart transplantation and azathioprine treatment, the mean activity was found to be 17.2 nmol/h/ml of RBC. We suggest screening for TPMT activity in transplant patients with leukopenia under AZA therapy. If TPMT deficiency is present, the AZA dosage should be adjusted or alternative immunosuppressive regimens should be considered.

Polymorphic Thiopurine Methyltransferase in Erythrocytes Is Indicative ofActivity in Leukemic Blasts From Children With AcuteLymphoblastic Leukemia

The activity of thiopurine methyltransferase (TPMT) exhibits genetic polymorphism, with approximately 1 in 300 individuals inheriting TPMT deficiency as an autosomal recessive trait, and about 11% having intermediate activity (ie, heterozygotes). Patients with TPMT deficiency accumulate excessive concentrations of 6-thioguanine nucleotides (TGNs) and develop severe toxicity when treated with standard dosages of mercaptopurine. High TPMT activity has been associated with lower concentrations of TGNs, yielding a higher risk of treatment failure in children with acute lymphoblastic leukemia (ALL). As the biochemical basis of these pharmacodynamic relationships has not been fully elucidated, we investigated the variability and relationship of TPMT activity in erythrocytes and lymphoblasts from children with ALL. A 58-fold range of erythrocyte TPMT activity was found among 119 patients receiving ALL chemotherapy (0.6 to 34.9 U/mL packed erythrocytes), but relatively low intrapatient variability (coefficient of variation, 13.5%) was observed over 1 year. A 27-fold range in TPMT activity was observed in leukemic blasts obtained from 42 patients at initial diagnosis (3.3 to 88.9 U/1 x 10(9) cells). TPMT activity in leukemic blasts at diagnosis was significantly correlated with TPMT in erythrocytes before therapy (rs = .75, P < .0001, N = 13). These data document extensive interpatient variability of TPMT activity in ALL blasts and establish its linkage to polymorphic TPMT activity in erythrocytes, providing a new mechanism by which erythrocytes serve as prognostic markers of mercaptopurine metabolism and TPMT activity in children with ALL.

Azathioprine-related bone marrow toxicity and low activities of purine enzymes in patients with rheumatoid arthritis

Azathioprine (AZA) metabolism largely parallels the endogenous purine pathways. To date, thiopurine methyltransferase (TPMT) deficiency has been reported as a cause of AZA-related bone marrow toxicity in 1 patient with rheumatoid arthritis (RA). We therefore studied purine enzyme activities in 3 patients with RA who experienced AZA-related bone marrow toxicity. Lymphocyte activity of purine nucleoside phosphorylase and 5'-nucleotidase (5NT) and erythrocyte activity of TPMT, key enzymes in thiopurine catabolism, were measured in 3 RA patients who had experienced AZA-related bone marrow toxicity and in 16 RA patients without signs of toxicity despite at least 6 months of treatment with AZA. Two patients with AZA-related bone marrow toxicity were found to have a TPMT deficiency, 1 partial and 1 total. In the third patient, 5NT activity was found to be well below the lowest level observed in the control subjects. All 3 patients with severe AZA-related bone marrow toxicity had abnormal purine enzyme activities. Deficiency of purine enzymes, including TPMT and 5NT, may be a cause of AZA-related bone marrow toxicity in patients with RA.

Thiopurine S-methyltransferase activity in a Chinese population.

Thiopurine S-methyltransferase is a cytosolic enzyme that catalyzes the S-methylation of thiopurine drugs. Although a genetic polymorphism has been recognized for this enzyme in populations of Caucasian descent, there has been scanty information about this polymorphism among Asians. In this study, we measured the erythrocyte thiopurine methyltransferase activity in 119 healthy Chinese subjects by a radiochemical assay. Methyltransferase activity was lower than what might have been expected for a white population. A bimodal frequency distribution was obtained that allowed the identification of four individuals with relatively low methyltransferase activity who may be heterozygotes for thiopurine S-methyltransferase deficiency; if so, the frequency of the mutant allele would be lower in this Chinese population than that observed in a white population (chi 2, p < 0.02). No gender-based differences were observed.

Altered mercaptopurine metabolism, toxic effects, and dosage requirement in a thiopurine methyltransferase-deficient child with acute lymphocytic leukemia

Thiopurine methyltransferase deficiency, inherited as an autosomal codominant trait, is associated with aberrant mercaptopurine metabolism leading to excessive cellular accumulation of 6-thioguanine nucleotides, the active metabolites of mercaptopurine. We describe a case of severe thiopurine methyltransferase deficiency (activity less than 1 U/8 x 10(8) erythrocytes) in a young girl with acute lymphocytic leukemia. The level of 6-thioguanine nucleotide in the patient's erythrocytes was seven times the population median value, and she had intolerable hematologic toxic effects during postremission therapy with a standard dosage of mercaptopurine (75 mg/m2 per day). Subsequent therapy with 6% of this dosage (10 mg/m2 three times weekly) yielded erythrocytic 6-thioguanine nucleotide concentrations consistently above the population median but not associated with prohibitively toxic effects. This case demonstrates that thiopurine methyltransferase deficiency does not absolutely contraindicate mercaptopurine therapy, and it also provides insight into the mechanism of excessive toxic effects of mercaptopurine sometimes observed in children with acute lymphocytic leukemia.