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| Journal of the New Zealand Medical Association,
25-February-2005, Vol 118 No 1210
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Measurement of thiopurine methyl transferase activity guides
dose-initiation and prevents toxicity from azathioprine
Christiaan Sies, Christopher Florkowski, Peter George,
Richard Gearry, Murray Barclay, James Harraway, Linda Pike, Trevor
Walmsley
Azathioprine, 6-mercaptopurine (6-MP), and thioguanine
are collectively known as thiopurines and are used in the treatment of
inflammatory bowel disease, acute lymphocytic leukaemia, myasthenia gravis,
autoimmune hepatitis, a variety of dermatological disorders, and as an
immunosuppressant in solid organ transplant
patients.1,2
Azathioprine is converted to
6-MP (Figure 1) which can then undergo one of three metabolic transformations.
The enzyme thiopurine methyl transferase (TPMT) has a trimodal variation in the
general population and is central to the understanding of the mechanisms of
toxicity associated with thiopurine drugs. Deficiency is inherited in an
autosomal recessive manner with 1 in 300 subjects having homozygous deficiency,
and around 8–10% of the community having intermediate enzyme activities.
Subjects with heterozygote deficiency will metabolise increased amounts of 6-MP
through the pathway to 6-thioguanine nucleotides (6-TGN). Although 6-TGN
production underlies the therapeutic action of azathioprine, excessive amounts
(as when TPMT is homozygous deficient) are extremely toxic and can result in
myelosuppression, neutropaenia, and potentially death.
Figure 1. The metabolism of 6-mercaptopurine via three
possible enzyme pathways: xanthine oxidase; hypoxanthine guanine
phosphoribosyltransferase (HGPRT); and thiopurine methyltransferase
(TPMT)
 There is considerable
potential to adjust thiopurine drug-starting doses with knowledge of TPMT
activity and to avoid potential myelotoxicity. We therefore undertook to
establish a routine assay for TPMT activity and establish appropriate reference
intervals and cut-offs for the New Zealand population.
MethodsTPMT activity is typically
measured in lysed red blood cells, which reflect the level of TPMT enzyme in
human liver, kidney, and normal lymphocytes.
A method for measuring erythrocyte TPMT activity has
been established based on the transfer of methyl groups from
S-adenosyl-L-[methyl-14C] methionine to
6-mercaptopurine, extraction of the radiolabelled
14C-methylated reaction product into isoamyl
alcohol/toluene, followed by liquid scintillation
counting.3,4
This assay has been used to assess the risk of possible
thiopurine toxicity in patients and to guide dose adjustments. Subjects with
lower enzyme activity (<12 U/ml RBCs) also underwent TPMT genotyping to
define the level of TPMT activity that separates true normals from those that
may be either normal or heterozygous deficient. The gene encoding TPMT is
located on chromosome 6 (6p22) and consists of 9 introns and 10 exons.
At least nine single-nucleotide polymorphisms (SNPs)
that lead to decreased TPMT activity have been identified in the coding
sequence. In addition, another SNP that leads to decreased TPMT enzymatic
activity has been identified at the intron IX/exon X splice
junction5. Testing for the common *2, *3A, and
*3C alleles was also undertaken, using a multiplexed allele-specific polymerase
chain reaction, which account for 90% of the known mutations in the Caucasian
population.
Figure 2. Histogram indicates the number of patients
confirmed as homozygous deficient (white); confirmed heterozygous (black);
normal genotype (grey); not genotyped (chequered) vs TPMT enzyme
activity
 ResultsOver
a 2-year period, 407 patients samples from throughout New Zealand were referred
to Canterbury Health Laboratories for TPMT phenotyping; these patients were
either about to receive azathioprine or had already started treatment. Their
TPMT activities are depicted in Figure 2, showing the expected trimodal
variation in the general population. Around 90% of the patient population had
normal to high activity with these usually having the wild type genotype
(*1/*1). Around 8% have reduced or intermediate activity, usually due to a
*1/*2,
*1/*3A, or
*1/*3C genotype. Those with either very
low or undetectable activity were found to be homozygous for the
*3/*3 mutation accounting for 0.7% of
the patient population.
ROC (Receiver operator
characteristic) curve analysis of the data shown in Figure 3 indicates that for
a patient with a TPMT activity of greater than or equal to 10.7 units/ml RBC,
the probability of a normal genotype is 100%.
Prior to the introduction of
the TPMT assay, a patient in Christchurch with inflammatory bowel disease was
commenced on azathioprine 1.5 mg/kg; 13 days later he developed severe
myelosuppression, with a haemoglobin of 61 g/L, WBC of 1.6, and neutrophils of
0.6; and required 3 days of barrier nursing and a 3 unit blood transfusion.
Subsequent TPMT testing revealed that this
patient was one of the 1 in 300 people who have no TPMT activity. Subsequently
two other homozygous deficient patients were identified by TPMT assay prior to
the initiation of therapy and avoided potential toxicity, with azathioprine
being introduced at 10% of the usual dosage.
Figure 3. Diagram of TPMT activity vs patients that are
heterozygous (1) or normal (0), with optimal cut-off defined by receiver
operator characteristic (ROC) analysis. The cut-off of 10.7 unit/ml RBC
identifies TPMT deficient subjects with 100% sensitivity
DiscussionWe have established a TPMT assay
in Canterbury Health Laboratories and used this with a view to prevention of
myelotoxicity and to guide initial azathioprine dosage.
The data presented correlates
well with similar studies on healthy populations, which show a 89:11:0.3 ratio
respectively.6 TPMT activity appears to show no
significant difference between the gender or age of the
patient.7 Thus the normal range of
9.3–17.6 unit/ml RBC, as defined in our population, can be used for
children as well as adults.
TPMT activity testing may be
useful for dose prediction and preventing toxicity. We identified three
homozygous deficient patients, one of which might have avoided complications had
the assay been undertaken prior to initiation of azathioprine. In those patients
with intermediate activity (i.e heterozygous deficiency), the thiopurine
starting dose should be lowered by
50–60%.8
Patients with Crohn’s
disease have been successfully treated with azathioprine using this regime even
when found to be TPMT deficient.9 Conversely,
for patients found to have significantly higher enzyme activity, a higher
starting dose may be appropriate and 6-TGN monitoring will also guide future
dose adjustment. Once initiated on thiopurines, future dose adjustments should
be guided by monitoring of 6-TGN levels, also available from our laboratory.
Regular monitoring of the white cell count should still be performed as toxicity
can still occur suddenly, often being induced by intercurrent illness or
initiation of additional
medications.10
Standard protocols for
treatment with thiopurines usually involve initial administration of low doses
followed by gradual increase. This is less than ideal, given that 90% of
patients may be receiving potentially inadequate treatment and the remaining 10%
may be exposed to potentially unnecessary toxic
effects.11 Although frequent monitoring of full
blood counts is usually in place for children with acute leukaemia other
patients (such as those with dermatological disorder) may go unnoticed until
myelosuppression has occurred.
Data for the year ending June
2002 shows that there were 14,203 prescriptions, dispensed on 37,105 occasions,
for azathioprine (personal correspondence with Pharmac). If one assumes that
these patients were on this drug for the full 12 months, this equates to about
3500 people on this drug. Over the last 5 years, there have been 35 reports of
adverse effects of azathioprine of which 5 had a haematological component (New
Zealand Centre for Adverse Reactions Monitoring (CARM), personal communication,
2004), which suggests that only a minority of adverse effects are
reported.
After review of our data, we
will perform confirmatory genotyping for the most common allelic variants on all
samples with activity of less than 12 units/ml RBC. To date, all subjects with
an activity above 10.7 units/ml RBC have normal genotypes, although below this
level there is a considerable overlap between heterozygous deficient and
genotypically normal subjects. Although some studies relate adverse effects
specifically to the genotype, it is probably more important to know enzyme
activity as genotypically normal subjects have a very broad range of enzyme
activities that may impact differently on the choice of starting
dose.12,13
Some national guidelines
already recommend that ‘azathioprine dose should be optimised both with
regard to efficacy and myelosuppression risk by prior measurement of thiopurine
methyltransferase (TPMT) activity.’14
Others have argued that it may be unethical, even legally culpable, not to
undertake some assessment of TPMT status prior to thiopurine drug
initiation.4
In addition to the
medicolegal risk, the cost of
treating neutropaenic patients is
considerable. Economic analysis has indicated that the prevention of
myelosuppresion in TPMT homozygotes, by screening each patient prior to
initiating treatment, has a favourable cost-benefit
ratio.15 In the UK, it has been stated that at
a cost of £40 for measurement of TPMT and with a frequency of 1 in 300 for
homozygous deficiency, £12,000 would be spent to prevent one fatal
toxicity16 and avert a proportion of toxicity
in heterozygous deficient individuals. Extrapolating the same figures to New
Zealand and with an assay cost of $57.75 per assay, this would equate to a cost
of $17,250 per fatal episode averted.
ConclusionThe combination of TPMT enzyme
activity measurement with genotyping allows for dose prediction and adjustment
to prevent dangerous side-effects in patients found to have lower than normal
activities.
Author information:
Christiaan Sies, Scientific Officer;
Christopher Florkowski, Chemical Pathologist; Peter George, Clinical
Director, Clinical Biochemistry Unit, Canterbury Health Laboratories,
Christchurch; Richard Gearry, Gastroenterology Research Fellow; Murray Barclay,
Gastroenterologist and Clinical Pharmacologist; James Harraway, Pathology
Registrar (Genetics); Linda Pike, Medical Laboratory Scientist; Trevor Walmsley,
Scientific Officer.
Correspondence:
Christiaan Sies, Clinical Biochemistry Unit, Canterbury Health Laboratories,
Christchurch. Fax: (03) 364 0320; email chris.sies@cdhb.govt.nz
References:
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