Molecular Diagnosis of Thiopurine S-Methyltransferase Deficiency: Genetic Basis for Azathioprine and Mercaptopurine Intolerance

Thiopurine S-methyltransferase (TPM) is a cytosolic enzyme that preferentially catalyzes the S-methylation (that is, inactivation) of such therapeutic agents as mercaptopurine, azathioprine, and thioguanine [1]. These thiopurine medications are currently used to treat many diseases, including cancer [2], autoimmune hepatitis [3], inflammatory bowel disease [4, 5], rheumatoid arthritis [6], multiple sclerosis [7], and autoimmune myasthenia gravis [8]; they are also used as immunosuppressants after organ transplantation [9, 10]. Several clinical studies have shown that patients with low TPM activity are at high risk for severe and potentially fatal hematopoietic toxicity if they are treated with conventional doses of mercaptopurine (for example, 75 mg/m2 body surface area per day) or azathioprine [9, 11-13]. Thiopurine S-methyltransferase activity shows codominant genetic polymorphism [14, 15]. About 90% of white and black persons have high TPM activity, and 10% have intermediate activity caused by heterozygosity at the TPM locus. About 1 in 300 persons inherits TPM deficiency as an autosomal recessive trait. Clinical studies have established an inverse correlation between TPM activity and accumulation of the active thioguanine nucleotide metabolites of mercaptopurine and azathioprine in erythrocytes. Patients with less efficient methylation of these thiopurine medications have more extensive conversion to active thioguanine nucleotides [2, 16]. Patients who have TPM deficiency accumulate higher levels of thioguanine nucleotides in erythrocytes if they receive standard doses of mercaptopurine or azathioprine. This accumulation of nucleotides usually leads to severe hematopoietic toxicity and possibly death [9], but this outcome can be averted if the thiopurine dose is decreased substantially (an 8- to 15-fold reduction) [17-19]. Patients who have intermediate TPM activity that is caused by heterozygosity at the TPM locus accumulate about 50% more thioguanine nucleotides than do patients who have high TPM activity [2]; this places patients with intermediate TPM activity at an intermediate risk for toxicity. Most of these patients are identified only after an episode of severe toxicity occurs. Although prospective measurement of erythrocyte TPM activity has been advocated by some investigators [4, 16], TPM assays are not widely available. Moreover, organ transplant recipients and patients who have recently received a diagnosis of cancer are frequently given transfusions of red blood cells; this precludes measurement of constitutive TPM activity before thiopurine therapy is started. Because thiopurine toxicity can be life threatening in TPM-deficient patients [9] and because of the intermediate risk for toxicity in heterozygous patients, a reliable method to identify patients who have inherited this trait is needed. If the genetic basis for TPM deficiency can be defined and polymerase chain reaction (PCR)-based methods can be developed to detect these inactivating mutations in genomic DNA, it should be possible to diagnose TPM deficiency and heterozygosity on the basis of genotype (as is now possible for other polymorphic enzymes) [17, 18]. To this end, we isolated and characterized two mutant alleles that are associated with TPM deficiency, TPM*2 and TPM*3A [19, 20]. The structures of these alleles are depicted in Figure 1. The molecular defect in TPM*2 is a G238C transversion mutation that leads to an amino acid substitution at codon 80 (Ala80Pro). Heterologous expression of this mutant allele in yeast showed a 100-fold decrease in S-methylation activity. The TPM*3A allele contains two nucleotide transition mutations (G460A and A719G) that lead to the amino acid substitutions Ala154Thr and Tyr240Cys. Heterologous expression of TPM*3A complementary DNA (cDNA) in yeast showed a greater than 200-fold reduction in TPM protein and undetectable activity. Moreover, marked instability of catalytic activity was evident for TPM proteins that were encoded by mutant cDNA containing either of these point mutations alone [20, 21]. We report the development, validation, and application of PCR-based methods for detection of these TPM mutations in the genomic DNA of patients and the elucidation of the polymorphic nature of the TPM gene locus in white persons. We also report a reliable method for the molecular diagnosis of TPM deficiency and heterozygosity that has excellent concordance between genotype and phenotype. Figure 1. Allelic variants at the human thiopurine S-methyltransferase (TPM) locus. Methods Human Patients and Determination of Phenotype Through methods described elsewhere [15], erythrocytes and leukocytes were isolated from the peripheral blood of healthy volunteers and children who had acute lymphoblastic leukemia. The volunteers were unselected blood donors who had been identified during a 2-month period, as described elsewhere [15]. The children were being treated at St. Jude Children's Research Hospital or had been referred for evaluation because they could not tolerate chemotherapy. Genotype was determined for all unrelated white patients who had TPM activity that indicated heterozygous or deficient genotypes and for the same number of unrelated persons who had high activity that indicated a homozygous wild-type genotype. We focused our initial studies on white patients because they belong to the ethnic group in which we have identified the largest number of TPM-deficient and heterozygous persons. The activity of TPM in erythrocytes was determined by the radiochemical assay of Weinshilboum and colleagues [22], whose methods we modified, as described elsewhere [15]. The TPM phenotype was assigned on the basis of TPM activity in erythrocytes and according to the criteria of Weinshilboum and Sladek (that is, patients who had <5.0 U/mL of packed red blood cells were considered TPM deficient, those who had 5 to 10 U/mL were considered heterozygous, and those who had >10 U/mL were considered homozygous wild-type) [14]. We used the lowest value of TPM activity in erythrocytes that was measured in each person. We extracted RNA from leukocytes by using the method of Chomczynski and Sacchi [23], and genomic DNA was isolated by chloroform-phenol extractions. The studies were approved by the institutional review board for clinical trials at St. Jude Children's Research Hospital, and informed consent was obtained from the patients or their guardians. Determination of Intronic Sequences The presence of a TPM-processed pseudogene [24] that could confound PCR-based genotyping methods and the absence of data on the genomic structure of the human TPM gene led us to initially use PCR primers that were complementary to TPM exon sequences to amplify genomic DNA by Expand PCR (Boehringer Mannheim, Indianapolis, Indiana) and thereby identify intronic sequences in the human TPM gene. The final volume for all PCR assays was 50 micro L. Through use of 1 g of placental genomic DNA (Clontech Laboratories, Inc., Palo Alto, California) as a template, PCR was done with primers A (5-GAGTTCTTCGGGGAACATTTCATTG-3) and B (5-CACCTGGATTAATGGCAAC TAATGC-3) in buffer D (Invitrogen, San Diego, California). The buffer contained Tris hydrochloride (pH 8.5), 60 mmol/L; ammonium sulfate, 15 mmol/L; and magnesium chloride, 3.5 mmol/L. The primers had been developed to amplify a fragment of genomic DNA (which included nucleotide 460) for detection of the G460A mutation. The concentration of each oligonucleotide was 0.1 OU/mL (about 0.5 mol/L), and 0.2 L Taq polymerase (Perkin Elmer Cetus, Norwalk, Connecticut) was used. With a Hybaid OmniGene thermocycler (Woodbridge, New Jersey), amplification was done for 30 cycles consisting of denaturation at 94 C for 1 minute, annealing at 55 C for 2 minutes, and extension at 72 C for 1 minute. A final extension step at 72 C for 7 minutes was also done. For the initial cycle, 5 L of deoxynucleoside triphosphates (dNTP, 10 mmol/L) was added after the temperature reached 80 C (following the hot start protocol). An amplified fragment of 138 base pairs was anticipated in the absence of intron sequences; the resulting fragment of 746 base pairs showed the presence of an intervening intron. This fragment was directly cloned into the plasmid pCR-II (Invitrogen). The recombinant plasmid was purified with Qiagen plasmid kits (Chatsworth, California) and sequenced with an automated sequencer using the cycle sequencing reaction and fluorescence-tagged dye terminators (Prism, Applied Biosystems, Foster City, California). The resulting intron sequence and the intron-exon boundary was then used to develop intron-specific primer P460F. Through a similar strategy, Expand PCR was used to amplify intron sequences that flanked the exons containing the G238C mutation (intron 4) and the A719G mutation (intron 9). The resulting intron-containing fragments were directly cloned into the plasmid pCR-II; the plasmid was purified and sequenced as described above. These sequences permitted the development of intron-specific PCR primers P2C and P719F for the detection of G238C and A719G mutations. Detection of TPM Mutations by Polymerase Chain Reaction Detection of G238C We used PCR amplification to determine whether the G238C transversion was present at the TPM locus. Genomic DNA, 400 ng, was amplified under conditions similar to those discussed for the intronic sequence except that 2 L of primer P2W (5-GTATGATTTTAT GCAGGTTTG-3) or P2M (5-GTATGATTTTATGCAGGTTTC-3) was used with primer P2C (5-TAAATAGGAACCATCGGACAC-3) (0.1 OU/mL) in each amplification. Unpurified PCR products were analyzed by electrophoresis in 2.5% MetaPhor gels (MetaPhor Agarose, FMC Bioproducts, Rockland, Maine) stained with ethidium bromide. A DNA fragment was amplified with P2M and P2C primers when C238 (mutant) was present, whereas a DNA fragment was amplified with P2W and P2C primers when G238 (wild-type) was present (Figure 2). Figure 2. Schematic of polymerase chain reac