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

Charles R. Yates, BS; Eugene Y. Krynetski, PhD; Thrina Loennechen, PhD; Michael Y. Fessing, PhD; Hung-Liang Tai, PhD; Ching-Hon Pui, MD; Mary V. Relling, PharmD; and William E. Evans, PharmD
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For author affiliations and current author addresses, see end of text. Acknowledgments: The authors thank Amy E. Atkinson, Linh Nguyen, and YaQin Chu for technical assistance; Sheri Ring, Margaret Edwards, and Lisa Walters for collecting blood samples; Dr. Howard McLeod for contributions to the phenotyping studies; Dr. Clayton W. Naeve of the SJCRH Biotechnology Resource Center; Drs. Mark Roberts, Denis R. Miller, Nora R. Rogers, D.J. Murry, and Gaston K. Rivera for their clinical acumen in the recognition and treatment of TPM-deficient patients; Dr. J. Boyett for guidance in the statistical analyses; and the patients and family members who participated in the study. Grant Support: In part by grant R37 CA36401, Leukemia Program Project grant CA20180, and Cancer Center CORE grant CA21765 from the National Institutes of Health; by a Center of Excellence grant from the State of Tennessee; and by American Lebanese Syrian Associated Charities. Requests for Reprints: William E. Evans, PharmD, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794. Current Author Addresses: Mr. Yates and Drs. Krynetski, Fessing, Tai, Relling, and Evans: Pharmaceutical Sciences, Thomas Tower, Room 1052, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105.

Copyright ©2004 by the American College of Physicians

Ann Intern Med. 1997;126(8):608-614. doi:10.7326/0003-4819-126-8-199704150-00003
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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.


Grahic Jump Location
Figure 1.
Allelic variants at the human thiopurine S-methyltransferase (TPM) locus.

Boxes depict exons in the human TPM gene. White boxes are untranslated regions, and black boxes represent exons in the open reading frame. Hatched boxes represent exons that contain mutations that result in changes to amino acids; these are the three point mutations detected by the genotyping methods using polymerase chain reaction.

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Grahic Jump Location
Figure 2.
Schematic of polymerase chain reaction (PCR)-based methods to detect G238C (top), G460A (middle), and A719G (bottom) mutations at the human thiopurine S-methyltransferase (TPM) gene locus.

For the sequence of PCR primers (P2W, P2M, P2C, P460F, P460R, P719F, P719R) and amplification conditions, see Methods. The bottom section of each panel shows an ethidium bromide-stained gel that depicts amplified DNA fragments for each of the genotype and PCR conditions described. bp = base pairs; Mut = mutant; Wt = wild type.

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Grahic Jump Location
Figure 3.
Thiopurine S-methyltransferase (TPM) activity in patients with different TPM genotypes determined by mutation-specific polymerase chain reaction methods.

The heavily shaded area depicts the range of TPM activity in erythrocytes that defines TPM deficiency (<5 U/mL of packed red blood cells), the lightly shaded area depicts intermediate activity that defines TPM heterozygous phenotypes (5 to 10 U/mL of packed red blood cells), and the unshaded area depicts the range of TPM activity in patients who have homozygous wild-type phenotypes. Black circles indicate patients with concordant genotype and phenotype; the black square indicates one patient with discordant genotype and phenotype.

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