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

Background: Thiopurine S-methyltransferase (TPM) catalyzes the S-methylation (that is, inactivation) of mercaptopurine, azathioprine, and thioguanine and exhibits genetic polymorphism. About 10% of patients have intermediate TPM activity because of heterozygosity, and about 1 in 300 inherit TPM deficiency as an autosomal recessive trait. If they receive standard doses of thiopurine medications (for example, 75 mg/m² body surface area per day), TPM-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 TPM deficiency and heterozygosity.

Design: Diagnostic test evaluation.

Setting: Research hospital.

Patients: The TPM phenotype was determined in 282 unrelated white persons, and TPM genotype was determined in all persons who had intermediate TPM activity (heterozygotes) and a randomly selected, equal number of persons who had high activity. In addition, genotype was determined in 6 TPM-deficient patients.

Measurements: Polymerase chain reaction (PCR) assays were developed to detect the G238C transversion in TPM*2 and the G460A and A719G transitions in TPM*3 alleles. Radiochemical assay was used to measure TPM activity. Mutations of TPM were identified in genomic DNA, and the concordance of TPM genotype and phenotype was determined.

Results: 21 patients who had a heterozygous phenotype were identified (7.4% of sample [95% CI, 4.7% to 11.2%]). TPM*3A was the most prevalent mutant allele (18 of 21 mutant alleles in heterozygotes; 85%); TPM*2 and TPM*3C were more rare (about 5% each). All 6 patients who had TPM deficiency had two mutant alleles, 20 of 21 patients (95% [CI, 76% to 99.9%]) who had intermediate TPM activity had one mutant allele, and 21 of 21 patients (100% [CI, 83% to 100%]) who had high activity had no known TPM mutation. Detection of TPM mutations in genomic DNA by PCR coincided perfectly with genotypes detected by complementary DNA sequencing.

Conclusions: The major inactivating mutations at the human TPM 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 TPM mutations provides a molecular diagnostic method for prospectively identifying TPM-deficient and heterozygous patients.