Thiopurine Methyltransferase Testing - TPMT


Indications for Testing

  • Patients being considered for thiopurine therapy in order to
    • Detect risk for severe myelosuppression with standard full dosing of thiopurine drugs
    • Individualize dosing of thiopurine drugs
  • Patients who have experienced an adverse reaction to thiopurine therapy

Laboratory Testing

  • TPMT genotyping
    • Detects *2, *3A, *3B, and *3C alleles
    • Variant alleles are correlated with risk of severe myelosuppression with standard dosing of thiopurine drugs
    • Does not replace need for clinical monitoring
  • Pretherapeutic or posttherapeutic use
    • Detect common genotypes
    • Detect enzyme phenotype
  • Should not be used in patients with history of allogeneic bone marrow transplant
  • TPMT phenotyping (enzyme activity) in red blood cells (RBCs)

    Low TPMT activity

    Normal TPMT activity

    High TPMT activity

    Units/milliliter (U/mL)




    Associated with

    High risk of bone marrow toxicity

    Low risk of bone marrow toxicity

    Therapeutic failure

    Dose adjustment suggested

    Dramatic dose reduction (80-90%) may be required


    Higher than standard dosing may be required


    Therapeutic drug monitoring may help optimize dose

    Represents 20th-99th percentiles

    Not well-characterized

    Limitations – may be inaccurate if performed within 30 days of erythrocyte transfer


  • Therapeutic drug monitoring (metabolic phenotype)
    • Thiopurine drug metabolites
      • Thiopurine metabolite concentrations may be appropriate for individuals with deficient or high TPMT activity to monitor and optimize dose
      • Metabolites detected


        Therapeutic Range

        Results Interpretation

        6-TGN RBC

        230-400 pmol/8 x 108 RBC

        <230 pmol/8 x 108 RBC – may indicate a reduced response to therapy

        >400 pmol/8 x 108 RBC – may indicate a higher risk for leukopenia

        6-MMPN RBC

        <5700 pmol/8 x 108 RBC

        >5700 pmol/8 x 108 RBC --  may indicate a higher risk for hepatotoxicity

Clinical Background

Thiopurine drugs such as azathioprine (AZA), 6-mercaptopurine (6-MP), and 6-thioguanine (6-TG) are widely used in the treatment of acute lymphoblastic leukemia (ALL), autoimmune diseases, inflammatory bowel disease, and posttransplant organ rejection. Patients with abnormal thiopurine methyltransferase (TPMT) enzyme activity due to genetics and/or drug-drug interactions have an increased risk of toxicity when given thiopurines.


  • Prevalence of phenotype
    • Low thiopurine methyltransferase (TPMT) activity – ~0.3%
    • Intermediate TPMT activity – ~10%
    • Normal TPMT activity – ~90%
    • High TPMT activity – unknown
  • Allele frequency by ethnicity

    Allele Frequency by Ethnicity


    TPMT *2

    TPMT *3A

    TPMT *3B

    TPMT *3C


























    Middle Eastern






  • TPMT gene – autosomal co-dominant inheritance
  • >20 TPMT deficiency alleles identified to date
    • TPMT deficiency alleles account for 95% of low-to-intermediate activity states
      • *2 (c.238G>C; p.Ala80Pro)
      • *3A (c.[460G>A;719A>G]; p.[Ala154Thr;Tyr240Cys])
      • *3B (c.460G>A; p.Ala154Thr)
      • *3C (c.719A>G; p.Tyr240Cys)
    • Homozygous or compound heterozygous
      • Associated with very low/no TPMT enzyme activity and high risk for drug-related toxicity with conventional thiopurine doses
    • Heterozygous
      • Associated with intermediate TPMT enzyme activity and increased risk for drug-related toxicity with conventional thiopurine doses
    • No variants detected
      • Predictive of *1 functional alleles
      • Predicts normal TPMT enzyme activity and normal risk for thiopurine drug-related toxicity


  • AZA, 6-MP, and 6-TG are inactive prodrugs used to treat a variety of different disease states and are metabolized by three different enzymes into three different 6-thioguanine nucleotides for activity
    • AZA is metabolized to 6-MP (one of most commonly prescribed)
      • 6-MP is converted into two pharmacologically inactive metabolites
        • 6-thiouric acid by the enzyme xanthine oxidase (XO)
        • 6-methylmercaptopurine (6-MMP) by thiopurine S-methyltransferase (TPMT)
          • Primary metabolic route for inactivation of nucleotides is catalyzed by TPMT
      • 6-MP is also converted into active thioguanine nucleotides by the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT) to exert therapeutic cytotoxic effects
      • When 6-MP is converted to inactive metabolites, the amount of 6-TG nucleotides reduces, which balances the amount of 6-TG nucleotides needed to achieve required cytotoxicity for therapeutic treatment
      • When TPMT enzyme activity is low, proportionately more 6-MP is converted into cytotoxic 6-TG nucleotides, which increases risk for toxicity
    • Accumulation of excessive nucleotides inhibit purine synthesis, most dramatically noted in the bone marrow, inhibiting cell proliferation and contributing to myelosuppression
    • Reduced drug dosing may prevent myelosuppression in patients with intermediate and low TPMT activity
      • Individuals with very low/no TPMT enzyme activity typically experience severe myelosuppression when receiving conventional thiopurine doses
      • An estimated 30-60% of individuals with intermediate TPMT activity who receive conventional thiopurine doses experience moderate to severe myelosuppression
    • Thiopurine dosing guidelines (Clinical Pharmacogenetics Implementation Consortium [CPIC])
    • TPMT can be inhibited by common drugs
      • NSAIDs
        • Ibuprofen
        • Ketoprofen
        • Naproxen
        • Mefenamic acid
      • Diuretics
        • Furosemide
        • Thiazides
      • Ulcerative colitis drugs
        • Sulfasalazine
        • Mesalamine
        • Olsalazine
  • Factors to consider if deciding when TPMT should be ordered
    • Disease state being tested
    • Starting dose
    • Need for immediate full dose
    • Previous documented tolerance of thiopurine medication at steady state doses

Indications for Laboratory Testing

  • Tests generally appear in the order most useful for common clinical situations
  • Click on number for test-specific information in the ARUP Laboratory Test Directory
Test Name and Number Recommended Use Limitations Follow Up
Thiopurine Methyltransferase, RBC 0092066
Method: Enzymatic/Quantitative Liquid Chromatography-Tandem Mass Spectrometry

Preferred screening test for pretherapeutic evaluation

Detects risk for severe myelosuppression with standard dosing of thiopurine drugs

Assesses risk prior to treatment with thiopurine drugs

Testing is contraindicated in patients who are currently administered thiopurine drugs prior to initiation of treatment

Does not replace clinical monitoring

Genotype cannot be inferred from TPMT activity (phenotype)

TPMT inhibitors may contribute to false-low test results

Blood transfusion within 30 days will reflect donor status

Thiopurine Methyltransferase (TPMT) Genotyping, 4 Variants 2012233
Method: Polymerase Chain Reaction/Fluorescence Monitoring

Assess risk due to genetics for severe myelosuppression with standard dosing of thiopurine drugs

Appropriate for pre- or posttherapeutic assessments

Consider if erythrocyte TPMT activity is abnormal or if such assessment not possible due to recent heterologous blood transfusion

Only targeted TPMT allele variants  will be detected by this panel

Diagnostic errors can occur due to rare sequence variations

Genotyping cannot distinguish between the *1/*3A and *3B/*3C genotypes

Genotyping does not replace the need for therapeutic drug monitoring or clinical observation

Genotyping in patients who have received allogenic stem cell/bone marrow transplant  will reflect donor status

Thiopurine drug metabolism and risk for toxicity may be affected by genetic and nongenetic factors that are not evaluated by this test

Test does not assess for TPMT allele variants associated with ultra-high enzyme activity

Thiopurine Drug Metabolites 2011134
Method: Quantitative Liquid Chromatography/Tandem Mass Spectrometry

Optimize thiopurine drug dosing for patients undergoing thiopurine therapy

Identify thiopurine metabolite concentrations that may lead to toxicity

Limit of quantification (LOQ)

  • LOQ – 12.5 pmol/8 x 108 RBC (6-TGN)
  • LOQ – 325 pmol/ 8 x 108 RBC (6-methyl mercaptopurine nucleotide [6-MMPN])