Stuart C. Pritchard

Thiopurine Methyltransferase Genotype Predicts Therapy-Limiting Severe Toxicity from Azathioprine

Azathioprine has demonstrated disease-modifying activity in rheumatic disease and is an effective therapy for inflammatory bowel disease, multiple sclerosis, systemic vasculitis, chronic hepatitis, dermatologic disorders, and immunosuppression after solid-organ transplantation [1-6]. However, its use has been limited by a high incidence of hematologic toxicity and concern about long-term side effects [7]. Azathioprine is a relatively inexpensive therapeutic agent for common diseases, and methods for prospectively identifying patients at risk for acute toxicity may be useful for therapeutic decision making. Azathioprine is converted to 6-mercaptopurine in vivo, where it is metabolized to cytotoxic thioguanine nucleotides or is inactivated by xanthine oxidase or thiopurine methyltransferase. Thiopurine methyltransferase-catalyzed S-methylation shunts thiopurine to relatively inactive compounds and away from activation to thioguanine nucleotides [7]. Population studies have found activity of thiopurine methyltransferase in erythrocytes to be trimodal: Approximately 90% of persons have high activity, 10% have intermediate activity, and 0.3% have low or no activity [8]. Azathioprine-induced bone marrow suppression was more frequent in patients with a dermatologic condition and the intermediate phenotype, whereas patients with high thiopurine methyltransferase activity had a poor clinical response [6]. Patients with low thiopurine methyltransferase activity have severe or fatal hematopoietic toxicity in response to thiopurine-based therapies [7, 9]. However, azathioprine also induces activity of thiopurine methyltransferase in erythrocytes, making direct assessment of enzyme activity difficult [10]. The recent identification of three distinct thiopurine methyltransferase mutations (detected in 80% to 95% of white persons with low or intermediate thiopurine methyltransferase activity) has allowed the development of polymerase chain reaction (PCR)-based techniques for genotype analysis [11]. The most common variant allele in white persons contains point mutations at nucleotides 460 and 719 and has been named TPM*3A [11]. Alleles containing a mutation at nucleotide 238 are designated TPM*2 [11]. This discovery led to the hypothesis that molecular analysis of thiopurine methyltransferase may be a useful way to identify patients at risk for toxicity from thiopurine medication. Methods Patients Thiopurine methyltransferase genotype was assessed in consecutive patients who were seen at the rheumatology departments of the Aberdeen Royal Infirmary or the Glasgow Royal Infirmary in Glasgow, United Kingdom, over a 6-month period and were prescribed azathioprine. No patients were excluded on the basis of previous or current medication. Patients received azathioprine, 2 to 3 mg/kg of body weight per day, as second-line therapy; some patients were also prescribed oral corticosteroids. The patients had received stable doses of nonsteroidal anti-inflammatory drugs as first-line therapy. Blood counts and liver function test results were regularly monitored at least monthly. Reasons for discontinuation of azathioprine therapy were intolerance, progressively abnormal liver function test results, and reduction in total leukocyte count to less than 3.5 109/L or reduction in neutrophil count to less than 1.5 109/L. Clinicians caring for the patients were unaware of the thiopurine methyltransferase genotype. Analysis of Thiopurine Methyltransferase Genotype After patients gave written informed consent, genomic DNA was extracted from 5 mL of whole blood and was analyzed for the presence of mutations at nucleotides 238, 460, and 719 by using allele-specific PCR or PCR restriction fragment length polymorphism, as described elsewhere [11, 12]. In brief, mutation-specific PCR was performed by using pairs of oligonucleotides that specifically amplified a PCR product in the presence of guanine (wild-type allele) or cytosine (mutant allele) at nucleotide 238 [11, 12]. Diagnostic assays for the other alleles took advantage of alterations in the presence of a restriction enzyme cut-site when mutations occurred at nucleotides 460 or 719 [11, 12]. Molecular analysis was done without knowledge of clinical outcome, and both positive and negative (no DNA) controls were included in each assay. Statistical Analysis Differences in the duration of therapy between patients with wild-type alleles and those with mutant alleles was assessed by the log-rank test, accompanied by Kaplan-Meier curves. Results All 67 patients recruited for this study (55 women and 12 men; mean age SD, 51 13.4 years) were evaluable for toxicity and duration of therapy. Azathioprine was used for rheumatoid arthritis (n = 49), systemic lupus erythematosus (n = 7), or other rheumatic diseases (n = 11). Therapy was discontinued in 25 patients (37%) because of side effects (nausea, abnormal liver function test results, and low leukocyte count) and in 18 patients (27%) because of lack or loss of efficacy. Six patients (9%) were heterozygous for TPM*3A; TPM*2 was not detected in this study sample. No patients were homozygous for low-activity thiopurine methyltransferase alleles. Of the 6 patients heterozygous for TPM*3A, 5 discontinued azathioprine therapy within 1 month of starting this therapy because of reduced total leukocyte counts (range, 0.9 to 2.7 109/L) (Figure 1). The sixth patient had a well-documented history of noncompliance with drug therapy and on subsequent questioning was found not to be taking azathioprine. Figure 1. Influence of thiopurine methyltransferase genotype on the duration of azathioprine therapy (61 patients had the wild-type TPM allele and 5 patients had the mutant TPM allele). Duration of therapy was significantly longer for patients with wild-type thiopurine methyltransferase alleles (median, 39 weeks [range, 6 to 180 weeks]) than for those with mutant alleles (median, 2 weeks [range, 2 to 4 weeks]) (P = 0.018). Within the first 2 months of therapy, liver function test results were abnormal in 6 patients with wild-type thiopurine methyltransferase alleles and 1 patient with mutant alleles. All other side effects were mild; the most frequent side effect was gastrointestinal upset. No hematologic abnormalities occurred in patients with wild-type alleles. Discussion Our study provides the first analysis of thiopurine methyltransferase genotype in patients with rheumatic disease and identifies the incidence of heterozygous genotype in this population. The clinical significance of this finding is shown in Figure 1: All patients with mutant thiopurine methyltransferase alleles were forced to discontinue azathioprine therapy within 1 month of initiating therapy because of serious hematologic side effects. The rate of abnormal liver function test results caused by azathioprine therapy was not associated with mutant alleles. These data strongly suggest that prospective analysis of thiopurine methyltransferase genotype may allow pretherapy identification of patients at risk for serious toxicity from azathioprine. Azathioprine therapy is active in patients with various inflammatory disorders and may still be useful in patients with variant thiopurine methyltransferase genotypes. However, substantial reductions in azathioprine dosage would be required [7]. Polymerase chain reaction-based assays are routinely used in many laboratory medicine departments; this availability facilitates the use of molecular therapeutic tests, such as thiopurine methyltransferase genotyping. Use of this approach may be appropriate from a health economic standpoint; at our center, for example, the cost of blood monitoring and supportive care exceeds that of PCR analysis by several-fold. This suggests that analysis of all azathioprine recipients to identify the 10% of the population at risk for thiopurine methyltransferase-mediated toxicity would be cost-effective. However, each individual center would need to verify this. Our study shows the ability of molecular analysis of thiopurine methyltransferase to identify patients at risk for hematologic toxicity from azathioprine. The use of such a test to avoid harmful, and potentially fatal, side effects is especially important because azathioprine is primarily used in ambulatory patients who do not have a terminal illness. Genotype analysis of thiopurine methyltransferase does not consider other variables regulating in vivo azathioprine activity, such as activation to thiopurine nucleotides, inactivation by xanthine oxidase, or dephosphorylation by 5-nucleotidase [13]. Assessment of the activity of thiopurine methyltransferase in erythrocytes may also prove useful. This testing will identify patients with high thiopurine methyltransferase activity who are resistant to therapy because thiopurine is shunted away from activation to thioguanine nucleotides [6, 14]. Alternatively, thioguanine nucleotides could be directly measured to determine the quantity of active metabolites formed, thereby predicting the dose necessary for clinical effectiveness [7]. However, only a few research centers have assays for enzyme activity or thioguanine nucleotides; this limits the widespread application of these assays as a screening test for azathioprine. Analysis of thiopurine methyltransferase genotype is a quick and relatively inexpensive way to identify patients at risk for acute toxicity from azathioprine and should also be applicable to related thiopurine drugs, such as 6-mercaptopurine. It may be a helpful tool for the clinical management of the many patients treated with azathioprine for inflammatory disorders. Drs. McLeod, Collie-Duguid, and Reid, Mr. Powrie, Mr. Matowe, and Mr. Pritchard: Department of Medicine & Therapeutics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. Dr. Capell: Centre for Rheumatic Disease, Glasgow Royal Infirmary, Glasgow G31 2ER, United Kingdom.

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.

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.

Analysis of thiopurine methyltransferase variant alleles in childhood acute lymphoblastic leukaemia

The role of 6‐mercaptopurine (6MP) in the treatment of childhood acute lymphoblastic leukaemia (ALL) is well established. However, the efficacy of 6MP is significantly influenced by inactivation by the polymorphic enzyme thiopurine methyltransferase (TPMT). In the general population 89–94% have high TPMT activity, 6–11% have intermediate activity, and approximately 0.3% have low activity. Individuals with low‐activity experience severe or fatal toxicity with standard 6MP doses. Prospective identification of this group of patients might prevent this problem.

CYTOCHROME P450 CYP3A5 IN THE HUMAN ANTERIOR PITUITARY GLAND

The cytochromes P450 are a key group of enzymes involved in the metabolism of xenobiotics and several biologically active endogenous compounds. The expression of CYP3A5 has been identified by reverse transcriptase‐polymerase chain reaction in human pituitary gland and shown by immunohistochemistry to be localized to growth hormone containing cells of the anterior pituitary gland. This is the first direct identification of an individual P450 subfamily in the pituitary gland and the presence of CYP3A in the pituitary gland may play a role in regulating growth hormone secretion.

Primary pulmonary osteosarcoma: case report and molecular analysis.

Primary pulmonary osteosarcoma is an extremely rare malignancy. To date, only 12 cases have been reported, with a high mortality rate. The authors report on a newly diagnosed patient and describe investigations that were performed using immunohistochemistry and comparative genomic hybridization (CGH).