Thiopurine S-methyltransferase genotypic analysis in autoimmune bullous diseases

ARTICLE

Auteur(s) : Maud Bezier¹, Ziad Reguiai¹, Fabien Vitry², Franck Broly³, Philippe Bernard¹

¹Department of Dermatology, Hôpital Robert-Debré, Centre Hospitalier de Reims, avenue du Général Koenig, 51092 Reims Cedex, France
²UAM, Hôpital Maison Blanche, Centre Hospitalier de Reims, France
³Laboratory of Biochemistry and Molecular Biology, Hôpital Calmette, Centre Hospitalier de Lille, France

accepté le 22 Avril 2008

Azathioprine is an immunosuppressive agent widely used in the prevention of organ transplant rejection and as a steroid-sparing agent in autoimmune diseases. The main indications of azathioprine in dermatology include different auto-immune bullous diseases, severe atopic dermatitis, chronic actinic dermatitis, systemic lupus erythematosus or vasculitis [1-3]. The major drawback of azathioprine therapy is the possible occurrence of myelosuppression (chronic leukopenia or acute bone marrow failure) which can be predicted by measuring thiopurine S-methyltransferase (TPMT) activity [4-6]. Other side-effects, including dose-dependent nausea, predisposition to infections and hypersensitivity reactions, are unrelated to TPMT activity. TPMT is a key enzyme in azathioprine metabolism, which has recently been more clearly defined and a mechanism for this acute toxicity has been identified [7]. Its activity is inversely related to the risk of developing severe hematopoietic toxicity in patients treated with standard doses of azathioprine. The cytosolic enzyme TPMT shows in vivo inter-individual and inter-ethnic variability caused by TPMT gene polymorphism, which is inherited as an autosomal co-dominant trait [8, 9]. Multiple alleles of the TPMT gene have been identified and three major inactivating mutations causing TPMT deficiency (G238C, G460A et A719G in exons 5, 7, and 10, respectively) either isolated or combined, have been identified in a European population and account for 80-95% of abnormalities of enzymatic activity [10-12]; 89% of Caucasians are homozygous for a functional allele of the TPMT gene, 11% are heterozygous for a functional allele of the gene and 1/300 individuals are compound heterozygous or homozygous for a non-functional allele of the gene. Three phenotypes correspond with these genotypes: high TPMT activity (high methylators), intermediate activity (intermediate methylators) and deficient activity (deficient methylators). This has led to the development of TPMT genotyping in order to identify patients at risk of azathioprine-induced myelosuppression [10]. The aim of the present study was to evaluate the usefulness of the TPMT genotyping in severe cases of autoimmune bullous diseases currently being or susceptible to be treated by azathioprine.

Patients and methods

Patients

TPMT genotyping

Data collection

An exhaustive list of all the TPMT genotypings performed between 1998 and 2006 for patients issued from our department (with autoimmune bullous diseases or other dermatological conditions) was also provided by the reference laboratory and the patients’ files analysed.

Results

A total of 75 patients with autoimmune bullous diseases (42 with BP, 15 with CP, 16 with pemphigus, 2 with EBA), who had been, at the time of diagnosis, believed susceptible to be further treated with azathioprine, had a TPMT genotyping. Among these, 70 patients (93%) had a high predictable TPMT activity and 5 (7%) an intermediate predictable activity (table 1). No patient showed a deficient TPMT activity. Only 34 out of 75 patients with an autoimmune bullous disease were further treated with azathioprine, this decision being made on the basis of either the severity or the cortico-resistance of the disease. TPMT genotyping was performed in 33 out of 34 patients (17 with BP, 6 with CP, 11 with pemphigus) who were currently being treated by azathioprine (mean initial dose: 1.7 mg/kg/day; mean maximal dose: 2.2 mg/kg/day; mean duration: 10 months). Thirty-one patients (94%) showed a high TPMT activity and two (6%) an intermediate TPMT activity (one patient heterozygous for a functional allele and a non-functional variant of the gene with G460A and A719G mutations; one patient with G238C mutation). A clinical complete remission was obtained in 14 out of 34 patients treated with azathioprine within a mean delay of 135 ± 85 days (mean ± 2 SD) and a mean maximal dose of 2.3 mg/kg/day (table 2). Azathioprine was associated with another treatment in 28 out of 34 patients (82%), including topical corticosteroids (n = 22), oral corticosteroids (n = 20) or both (n = 14), intravenous immunoglobulins (n = 5) or other therapies (dapsone, plasma exchange, tetracyclines; n = 4).

Adverse reactions were observed in 23 out of 34 patients (68%) leading to withdrawal of treatment in 17 patients (table 3). Among these, there were two cases (6%) of hypersensitivity reaction which occurred within a mean delay of 20 days. In one of these two patients, azathioprine was reintroduced with recurrence of identical symptoms within a shorter time duration (24 hours). Clinical signs during these hypersensitivity reactions included fever, arthralgias, skin rash, gastrointestinal symptoms, pancreatitis and hepatitis. Haematopoietic side-effects were observed in 12/34 patients (35%) treated with a mean maximal dosage of azathioprine of 2.7 mg/kg/day; they occurred over a mean period of 108 days, despite a high predictive TPMT activity in all affected patients. Although generally mild, these haematopoietic side-effects led to the interruption of azathioprine therapy in 8/12 patients. No myelotoxicity was observed in the two patients with intermediate TPMT activity (mean maximal azathioprine dosage: 1.7 mg/kg/day). Gastro-intestinal effects or infections were respectively reported for 8 and 10 patients.
Table 1 TPMT Genotyping in patients with autoimmune bullous diseases (n = 75) and other dermatologic disorders (n = 42)

²Heterozygous for a functional allele of the gene and nonfunctional variant of the gene with mutations G460A and A719G.

³Heterozygous for a functional allele of the gene and nonfunctional variant of the gene with mutation G238C.

⁴Heterozygous for a functional allele of the gene and nonfunctional variant of the gene with mutation A719G.

Table 2 Characteristics of patients treated with azathioprine with a further complete clinical remission

²Cicatricial Pemphigoid.

³Pemphigus.

⁴Weight not done.

Table 3 Adverse events in patients treated with azathioprine (AZA) (n = 23)

• Adverse events: leukopenia (10⁹/L) grade 1: 3.0-3.9, grade 2: 2.0-2.9, grade 3: 1.0-1.9
• neutropenia (10⁹/L), grade 1: 1.5-1.9, grade 2: 1.0-1.4, grade 3: 0.5-0.9
• anemia (g/L) grade 1: 95-109, grade 2: 80-94
• nausea and vomiting grade 1: nausea
• diarrhea grade 1: < 2 days, grade 2: >2 days no necessary medication
• infection grade 1: minor, grade 2: moderate, grade 3: major, grade 4: sepsis with hypotension
• cytolysis grade 1: 1.26-2.5 N, grade 2: 2.6-5 N.

Discussion

TPMT is the best studied enzyme involved in thiopurine metabolism and represents an interesting example of practical pharmacogenomics [7, 8]. After oral ingestion, azathioprine, an antimetabolite in the metabolism of purines, is rapidly converted into its active metabolite 6-mercaptopurine (6-MP) in vivo and metabolized by three competing enzymes. The two catabolic enzymes TPMT and xanthine oxidase (XO) produce inactive metabolites. The third enzyme, hypoxanthine-guanine phosphoribosyltransferase, converts azathioprine into 6-thioguanine nucleotides (6TGn) which are responsible for the cytoxicity via their incorporation into RNA and DNA [16]. Several clinical studies have found that high methylators may actually be insufficiently treated with conventional doses of azathioprine, whereas intermediate and deficient methylators are known to be at risk for moderate to severe hematopoietic toxicity when treated with standard doses [5, 7, 17]. This is due to an inverse relationship between TPMT activity and production of active metabolites of azathioprine (6TGn). Two techniques can be used to measure TPMT status: enzyme-level testing (phenotype testing) and DNA based testing (genotype testing). However, evaluation of the phenotypic status of patients by the measurement of red blood cell may be altered by environmental factors, especially in patients receiving blood transfusions or certain drugs that may interfere with TPMT metabolism, which may limit the usefulness of those phenotyping procedures. Unlike the previous series of Snow and Gibson [4], the TPMT genotyping technique used in our present study also allowed us to predict enzyme activity.

The recommended standard dosage of azathioprine for dermatological indications in order to be safe and efficient is 1-3 mg kg^–1 daily [1, 2, 21], adjusted within these limits according to the response, and care should be taken when prescribing azathioprine in the elderly.

From our present results based on TPMT genotype, a highly predictable TPMT activity authorizes azathioprine dosage superior to 2 mg/kg/day, though this is not an absolute guarantee against the occurrence of hematopoietic side-effects. Indeed, azathioprine-induced myelotoxicity could also be ascribed to non-functional TPMT gene mutations in 10% of cases in the European population [11], to factors such as viral infections or drug interferences (allopurinol, salicylates), or to other azathioprine metabolic pathway disturbances [17]. In a previous report of Spire-Vayron de la Moureyre et al. who used the same genotyping technique as in the present study, 15 out of 164 individual homozygotes for the functional allele actually presented a slightly decreased TPMT activity [11]. The hypothesis that rare and unknown non-functional alleles of the TPMT gene are not discovered so far is possible, but several non functional variants of the gene have been characterized and major mutations causing loss of function of alleles have been recognized [12, 18, 19]. Moreover, our elderly patients with high TPMT activity achieved a complete clinical remission with a mean maximal dosage (2.4 mg/kg/day) in accordance with previous report by Snow and Gibson [4]. Patients with intermediate TPMT activity require a substantial reduction in doses and careful management to avoid severe bone marrow toxicity [15]. In our series, only 2 out of 5 patients with an autoimmune bullous disease who had an intermediate predictable TPMT activity were actually treated with azathioprine at a moderate dosage, i.e. 1.5-2 mg per kg daily (table 2). In these two patients, no myelotoxicity was observed and the disease further remained controlled under lower dosages of azathioprine, illustrating that TPMT-based azathioprine dosage adjustment may be of clinical value.

Screening for TPMT measurement may also be a cost-attractive strategy. The direct cost of hospital treatment in the UK of an episode of azathioprine-related myelotoxicity was estimated at ₤3200 in 1997 [6]. A more recent cost analysis performed in Canada, where a single TPMT test costs CAN$100, suggested that TPMT screening was cost-neutral when considering only the prevention of myelosuppression in TPMT-deficient individuals (based on a population incidence of 1/300 TPMT deficiency) and cost-beneficial when considering the prevention of myelosuppression in patients with intermediate TPMT activity [20]. But in fact, the number of patients studied in our preliminary study did not allow us to detect the very rare individuals with completely deficient activity in whom azathioprine must be avoided. Determination of TPMT activity, in our view, may always be used in severe cases of BP, as in most cases of pemphigus, though the clinical use of azathioprine is decreasing in recent years in these two conditions. This is due to the emergence of new therapeutic strategies such as rituximab in pemphigus [22] or methotrexate in BP [23, 24] which are currently developed as alternatives to classical immunosuppressant drugs in France. However, apart from autoimmune bullous diseases, a number of indications for azathioprine remain in dermatological disorders, including severe cases of atopic dermatitis or chronic actinic dermatitis [1, 13-15, 21]. Since newer immunosuppressants are much more expensive and relatively unproven in many instances, azathioprine probably will remain in dermatology for years [1]. The use of TPMT testing to guide azathioprine prescription could rise in current practice with the increasing of clinical TPMT testing services and a better knowledge about the risks of prescribing azathioprine in patients with abnormal TPMT activity. In conclusion, although strongly recommended before azathioprine treatment [1, 2, 5, 6, 21, 25, 27, 28, 30, 32], predicting TPMT activity appears only marginally helpful in patients with autoimmune bullous diseases, due to the rarity of deficient methylators (one in 2-300 individuals in the general population). In clinical practice, its main interest is to adjust more precisely the azathioprine dosage in predicted high- and intermediate-methylators. However, a high predicted TPMT activity using genotyping is not a guarantee against the occurrence of haematological side-effects [26, 29, 31] since about 10% of homozygotes for the functional allele may actually present a slightly decreased TPMT activity [11] and a regular haematological monitoring during the treatment remains mandatory.
Table 4 Recent studies evaluating the relationship between TPMT activity and azathioprine (AZA) hematological side effects

Acknowledgements

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