Is thiopurine methyltransferase genetic polymorphism a major factor for withdrawal of azathioprine in rheumatoid arthritis patients?
H. Corominas,
M. Domènech1,
A. Laíz,
I. Gich2,
C. Geli,
C. Díaz,
F. de Cuevillas1,
M. Moreno,
G. Vázquez and
M. Baiget1,
Unitat de Reumatologia, Servei de Medicina Interna,
1 Servei de Genètica and
2 Servei Epidemiologia and Hospital Universitari de la Santa Creu i Sant Pau, Barcelona, Spain.
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Abstract
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Objective. To determine whether the presence of thiopurine methyltransferase (TPMT) alleles associated with reduced or absent activity of thiopurine methyltransferase is a major factor for withdrawal of azathioprine (AZA) in rheumatoid arthritis (RA) patients.
Methods. The TPMT genotype, including the variable number of tandem repeats (VNTR) pattern in the 5' untranslated region, was analysed in 111 patients with long-standing RA. Azathioprine (AZA) therapy was used in 40 patients (36%) as a disease-modifying anti-rheumatic drug.
Results. Seven out of 111 RA patients (6.3%) were carriers of a mutant allele, TPMT3A (G460
A, A719
G) being the mutant allele observed most frequently. In the group of 40 AZA-treated patients, therapy was discontinued in six patients because of side-effects and in 26 patients because of lack of efficacy. Three patients presented moderate side-effects and were homozygous for the wild-type TPMT allele, whereas the remaining three patients, who developed gastrointestinal effects with severe nausea and vomiting, were TPMT3A carriers
Conclusion. In this observational study, the absence of response, probably due to the low-dose scheme used, was the major cause of AZA withdrawal in our series of RA patients. TPMT genotyping may allow the use of high doses of AZA in patients with normal TPMT alleles to improve the efficacy of this immunosuppressive drug. Our data support the relationship between gastrointestinal intolerance and thiopurine metabolic imbalance.
KEY WORDS: Thiopurine S-methyltransferase (TPMT), Rheumatoid arthritis, Azathioprine.
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Introduction
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Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown aetiology, with chronic inflammation, joint pain and morning stiffness that leads to bone erosion [1]. Although immunosuppressive therapy with azathioprine (AZA) is widely used because of its recognized efficacy, withdrawal of the drug is sometimes required due to side-effects such as gastrointestinal effects [2] and bone marrow toxicity [3, 4]. This myelosuppressive toxicity seems to be related to an anomaly in the purine metabolic pathway resulting from deficient thiopurine S-methyltransferase (TPMT) activity [5].
TPMT (E.C.2.1.1.67) is an enzyme involved in the intracellular metabolism of mercaptopurine [6]. The immunosuppressant AZA is converted into 6-mercaptopurine (6-MP), which is metabolized by TPMT, oxidized by xanthine oxidase to thiouric acid or catabolized to 6-thioguanine nucleotides (TGN) by hypoxanthine phosphoribosyl transferase (Fig. 1
).

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FIG. 1. Scheme of thiopurine drug metabolism. XA, xanthine oxidase; HPRT, hypoxanthine phosphoribosyl transferase.
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In a random population of 298 subjects, Weinshilboum and Sladek [7] reported that 86.6% had high TPMT activity, 11.1% intermediate activity and 0.3% low activity. Thus, according to the level of TPMT activity, the population could be classified as at low, moderate or high risk of bone marrow toxicity.
TPMT activity is usually measured in red blood cells and reflects the enzymatic activity in human liver, kidney and leukaemic lymphoblasts [8, 9]. Clinical studies with 6-MP and AZA have established an inverse correlation between erythrocyte TPMT activity and the accumulation of 6-MP metabolites in erythrocytes [5, 10]. Patients with low TPMT activity accumulate higher levels of 6-TGN metabolites and are at a higher risk of potential bone marrow toxicity after receiving standard doses of 6-MP and AZA than those with high enzyme activity [11]. The use of erythrocyte 6-MP metabolite (TGN and 6-MMP) levels and TPMT activity has therefore proved clinically useful in drug monitoring [12].
The TPMT gene is located on chromosome 6p22.3, spans approximately 34 kilobases of genomic DNA and contains 10 exons. Tai et al. [13] identified two mutant TPMT alleles [460 (G
A) and 719 (A
G)] associated with loss of catalytic activity. Further studies have identified additional allellic variants that correlate with low enzymatic activity [14, 15]. Table 1
summarizes the allelic variants described to date. A polymorphic tandem repeat was recently identified within a GC-rich area in the 5'-flanking region of the human TPMT gene [16]. The repeat elements were either 17 or 18 base pairs in length and the most common alleles were those with either four (V4) or five (V5) repeats. As the level of TPMT activity was inversely related to the sum of the number of repeat elements on the two alleles in a given individual, the authors concluded that the VNTR modulated the level of TPMT activity.
We studied the TPMT genotype in a group of RA patients treated with AZA to elucidate the genetic basis of the adverse side-effects or lack of efficacy leading to the withdrawal of the drug.
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Patients and methods
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Subjects
One hundred and eleven non-selected out-patients with RA according to the American Rheumatism Association criteria [17] were included in this observational study. AZA therapy was used in 40 patients (36%) as a disease-modifying anti-rheumatic drug. Therapy was discontinued in six patients (15%) because of side-effects and in 26 patients (65%) owing to lack of efficacy, and eight patients (20%) currently remain under AZA therapy. All were unrelated white Caucasian subjects attending the Rheumatology Clinic in the Internal Medicine Department of Sant Pau University Hospital, Barcelona. TPMT genotyping had been performed previously in a control group of 105 Spanish healthy individuals [18].
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Methods
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Genomic DNA was extracted from peripheral blood nucleated cells according to Miller et al. [19]. All samples were analysed for the presence of mutations at nucleotides 460 and 719 using polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP), as described elsewhere [20]. Two selected groups of samples (from RA patients in whom AZA therapy had been discontinued because of side-effects and from those in which PCR-RFLP identified a mutant allele) were subsequently analysed by direct sequencing of exons 5, 7 and 10 to look for known mutations at nucleotides 238, 292, 644 and 681 in the coding region and to a G
A change in intron 9. Sequencing of exon 7 confirmed the results obtained with PCR-RFLP. DNA fragments were amplified by means of primer sequences described previously [21], using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions. Electrophoresis was carried out on an ABI Prism 377 DNA Sequencer (Perkin-Elmer Applied Biosystems). In these two groups of samples, the polymorphic variable number of tandem repeats (VNTR) located in the 5'-flanking region of the TPMT gene was typed using previously described primers [16] and the direct sequencing approach mentioned above.
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Results
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We evaluated genetic polymorphism in the TPMT gene in a large group of RA patients to elucidate the different clinical response after the introduction of AZA. The results were compared with those of a control group of healthy individuals of similar ethnic background [18].
In the healthy control group, 10/105 (9.5%) were heterozygous carriers of the mutant allele of the TPMT gene [18]. Within the RA patients, 7/111 (6.3%) were carriers of a mutant allele (Table 2
). Patient 1 was a double heterozygote for alleles TPMT3A and TPMT3B and patient 4 was heterozygous for the TPMT3B allele; both patients showed a V4/V4 genotype in the VNTR region. The remaining five patients were TPMT3A carriers. Their genotypes at the VNTR region were as follows: 2/5 were V4/V4 homozygotes, 2/5 were V5/V5 homozygotes and the remaining patient was V4/V5 (Table 2
). As in the control group, the TPMT3A (G460
A, A719
G) allele was the mutant allele observed most frequently.
Therapy was discontinued because of (i) side-effects (gastrointestinal 7.5%, abnormal liver function test 2.5%, slight decrease in leucocyte count 5%) in 6/40 patients (15%), (ii) lack of efficacy in 26/40 patients (65%) and (iii) the development of an infectious process unrelated to the AZA treatment in one patient. Eight individuals are still under AZA therapy (Table 3
).
The presence of side-effects was the reason for the withdrawal of AZA in six patients. Three of them (two with altered liver function and one with moderate leucopenia) were homozygous for the wild-type TPMT allele and were also homozygous V4/V4 in the VNTR region. The remaining three patients (patients 5, 6 and 7 in Table 2
), who presented gastrointestinal effects with severe nausea and vomiting with 1.0, 1.3 and 0.7 mg/kg of the drug respectively, were heterozygous carriers of allele TPMT3A. Two of them (patients 5 and 7 in Table 2
) were homozygous (V5/V5) in the VNTR region while patient 6 in Table 2
was heterozygous (V4/V5) in this region.
Table 2
summarizes the clinical effects of AZA in patients presenting genetic polymorphism of TPMT. Patients 1 and 2 did not take AZA. Patient 3, who received a higher dose of AZA (1.4 mg/kg), developed an infectious process and AZA was withdrawn. This patient was under AZA therapy for a short time (2 weeks) and, although being heterozygous for a mutant allele, he did not develop myelosuppression. Patient 4 did not develop bone marrow toxicity despite the presence of a mutant allele which could lead to impaired TPMT activity, because he received a small dose of AZA (0.8 mg/kg) to control the disease. Patients 5, 6 and 7 are discussed in the previous paragraph.
Direct sequencing of exons 5, 7 and 10 was performed in the DNA samples from RA patients in whom AZA therapy was discontinued because of side-effects and in those in whom PCR and restriction analysis identified a mutant allele. No changes at nucleotides 238, 292, 644 or 681 in the coding region or a G
A change in intron 9 were identified in these samples.
A 2x2 statistical analysis of patients with and without toxicity vs those with and without mutant genotypes showed a significant difference (Fisher's exact test, P=0.018). These data show that the positive predictive value for a carrier of a TPMT mutant allele is 60% for the development of AZA intolerance, with a sensitivity of 50% and a specificity of 94.1%.
Table 4
shows the frequencies of the TPMT mutant alleles found in samples of Spanish healthy controls [18] and in the three different groups of RA patients included in the study: group 1, RA patients without AZA therapy; group 2, RA patients under AZA therapy without side-effects; and group 3, RA patients under AZA therapy with side-effects. We examined if there was a significant difference between the genotype frequencies in the various groups (
2 test). RA patients under AZA therapy with side-effects (group 3) showed significant differences when compared with controls (P=0.021), with group 1 (P=0.003) and when compared with group 3 (P=0.018).
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TABLE 4. Frequencies of TPMT genotypes in the RA patients included in the study and in Spanish control individuals
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Discussion
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AZA is a second-line therapy used in the treatment of autoimmune rheumatic disorders such as RA. Patients treated with AZA present a varying response, ranging from clinical remission to an unsatisfactory response and severe toxicity. Long-term studies confirm that this immunosuppressive therapy must be withdrawn in 1030% of AZA-treated patients owing to side-effects [22].
The genetic polymorphisms of TPMT that condition enzymatic activity seem to play a major role in thiopurine drug-induced toxicity. Patients with low TPMT levels accumulate high levels of thiopurine nucleotides when treated with standard doses of purine-related drugs such as AZA and 6-MP. As this accumulation of nucleotides usually leads to severe haematopoietic toxicity, measurement of TPMT activity has been recommended before introducing these drugs. Measurement of TPMT activity is usually performed in erythrocyte lysates [9] but several methodological factors should be taken into account: (i) results can be masked by recent blood transfusions; (ii) the measurement is only available in specialized laboratories; (iii) a sample of 1020 ml of fresh whole blood is required; and (iv) samples cannot be kept for subsequent analysis.
New advances, recently reviewed by Coulthard and Hall [23], have led to the description and characterization of a series of single-nucleotide polymorphisms that result in low TPMT activity. The wild-type allele for high enzymatic activity has been designated TPMT1. Genotyping of individuals with intermediate and low TPMT activity has shown that the TPMT3A allele is the most prevalent mutant allele associated with TPMT deficiency in Caucasians [13]. In addition to the variant alleles associated with significantly decreased levels of TPMT activity, the number of repeat elements in the VNTR region located in the TPMT gene promoter could modulate levels of enzymatic activity. The most common alleles were those with four (V4) or five (V5) repeats [24].
In the last few years, the pharmacogenetics of TPMT has been studied in different groups of patients receiving 6-MP or AZA, and in most cases the TPMT genotype correlates well with in vivo enzyme activity in erythrocytes and is clearly associated with the risk of toxicity.
Stolk et al. [25] studied TPMT activity and the development of side-effects in 33 patients with RA and they concluded that inherited intermediate TPMT activity seems predictive of the development of severe AZA side-effects. Interestingly, they hypothesized that gastrointestinal intolerance might also be related to a thiopurine metabolic imbalance.
Black et al. [22] studied 67 patients treated with thiopurine drugs for rheumatic disease and identified six patients (9%) as heterozygous for mutant TPMT alleles. Five of these six patients discontinued therapy because of low leucocyte counts within 1 month of starting treatment and the sixth patient showed non-compliance with AZA therapy. Patients with wild-type TPMT alleles received therapy for a median of 39 weeks compared with a median of 2 weeks in patients heterozygous for mutant TPMT alleles. This study clearly demonstrated that prospective knowledge of the TPMT genotype would aid clinical management of patients undergoing thiopurine therapy.
In other autoimmune diseases, such as systemic lupus erythematosus, disease-related neutropenic episodes are difficult to distinguish from those caused by drugs. To address this issue, Naughton et al. [26] determined the TPMT genotype in patients taking AZA. They concluded that the presence of TPMT mutant alleles could predict a neutropenic episode but that not all neutropenias could be predicted by TPMT genotyping.
In the group of 111 RA patients in this study, seven (6.3%) were carriers of a mutant TPMT allele, a figure similar to that found in a group of Spanish control individuals [18].
In the RA patients included in this study, the major cause of AZA withdrawal was the lack of efficacy (26 of the 40 RA patients under treatment). This high percentage (65%) of patients not responding can be attributed to the low doses of AZA administered. In our hospital, the protocol for AZA treatment in RA patients begins with 0.5 mg/kg, and if no adverse effects are observed after 2 weeks of treatment, higher doses, of up to 11.5 mg/kg, are administered for 3 additional months. After this period, if no clinical improvement is achieved, AZA is withdrawn owing to lack of efficacy. TPMT genotyping performed before AZA therapy will allow us to identify the group of RA patients with normal TPMT alleles. We consider that treatment of these low-risk patients with high doses of AZA (up to 2 mg/kg) will improve the response to AZA therapy in RA patients.
In our series, six out of 40 patients (15%) presented mild to moderate side-effects. Two of the 40 presented a slight decrease in the total white blood cell count and one patient demonstrated abnormal liver function; these three patients were all homozygotes for the TPMT wild-type allele. Three cases with gastrointestinal adverse effects were heterozygous carriers of the TPMT3A allele. These data support those of Stolk et al. [25] in that gastrointestinal intolerance could also be related to a thiopurine metabolic imbalance.
AZA is used relatively seldom in the treatment of RA nowadays. Our observational study showed that the major cause of withdrawal of AZA in RA patients was the absence of response to the drug, probably because of the low-dose treatment regimen that was used in the patients studied. TPMT genotyping could allow the use of high doses of AZA in patients with normal TPMT alleles to improve the efficacy of this immunosuppressive drug.
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Acknowledgments
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We thank Antònia Cortès for her technical support in the management of samples.
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Notes
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Correspondence to: M. Baiget, Servei de Genètica, Hospital de la Santa Creu i Sant Pau, Avda Sant Antoni M. Claret 167, 08025 Barcelona, Catalonia, Spain. E-mail: mbaiget{at}hsp.santpau.es 
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Submitted 2 May 2002;
Accepted 28 May 2002