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

Background: Thiopurine 5-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/m(2) body surface area per day), TPMT-deficient patients accumulate excessive thioguanine nucleotides in hematopoietic tissues, which leads to severe and possibly fatal myelosuppression. Objective: To elucidate the genetic basis and develop molecular methods for the diagnosis of TPMT deficiency and heterozygosity. Design: Diagnostic test evaluation. Setting: Research hospital. Patients: 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. Measurements: Polymerase chain reaction (PCR) assays were developed to detect the G238C transversion in TPMT*Z 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. Results: 21 patients who had a heterozygous phenotype were identified (7.4% of sample [95% Cl, 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. Conclusions: 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.