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• Re: Is ITPA genotyping predictive for the development of side effects to AZA?
  Jolanda M van Dieren
  Published on: 16 January 2006
• Is ITPA genotyping predictive for the development of side effects to AZA?
  John A Duley
  Published on: 14 November 2005

• Published on: 16 January 2006

  Re: Is ITPA genotyping predictive for the development of side effects to AZA?

  • Jolanda M van Dieren, PhD student
  • Other Contributors:
    • Hanneke A.J. van Vuuren, Ernst J. Kuipers, Bettina E. Hansen, Johannes G. Kusters, Edward E.S. Nieuwenhuis, C. Janneke van der Woude

  Dear Editor,

  In response to the comments of Duley et al. to our letter published in GUT 2005 we would like to make some additional remarks.

  First of all, Duley et al. conclude that our study had a retrospective character, but this is not correct. This is a longitudinal cohort study in which all patients with IBD visiting our out-patient department were included. These patients were followed prospectively fo...

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  Dear Editor,

  In response to the comments of Duley et al. to our letter published in GUT 2005 we would like to make some additional remarks.

  First of all, Duley et al. conclude that our study had a retrospective character, but this is not correct. This is a longitudinal cohort study in which all patients with IBD visiting our out-patient department were included. These patients were followed prospectively for the development of AZA-associated side effects. TPMT and ITPA genotypes were determined during the follow-up period.

  Next, Duley et al question the quality of our PCR sensitivity for the detection of the A719G TPMT mutation. In contrast to what is implicated, we performed our genotyping of the A719G mutation in three different ways. First, we determined the digestion of the Acc1 site on the PCR product of genomic DNA. In one case with an adequate positive control, we found a homozygous G460A mutation, but no A719G mutation, thus representing a homozygous TPMT*3B polymorphism. This result was corroborated by both DNA sequencing and an INNO-LiPA assay in an independent laboratory.

  In relation to TMPT we appreciate that a homozygous TPMT polymorphism, leading to absence of enzymatic TPMT activity can lead to severe leukopenia as was the case in our study. Analysis of TPMT polymorphisms would therefore benefit the few (1/300) homozygote patients. However, we and others noted that heterozygous TPMT is not a significant predictor for AZA-related side effects. In addition, a high percentage of patients using AZA develop side-effects (10-20%), irrespective of their TPMT genotype. As a consequence determination of these genotypes has only limited impact on the prescription policy of AZA and the recommended follow-up of patients. Tests other than genotyping, such as measuring TPMT activity or AZA metabolites, may be more useful in monitoring AZA treatment. To gain consensus about this discussion a large cost- effectiveness study should be performed.

  In relation with ITPA, to our knowledge two studies have observed an association between heterozygous ITPA 94C>A polymorphism and adverse drug reactions.[1,2] The p-values presented in these studies were described as statistically significant. However, they were in the range that would require further studies before being generally accepted.[3] In fact, there are now 4 studies (and one upcoming) that demonstrate no significant correlation between and the ITPA 94C>A polymorphism and the development of adverse drug reactions on AZA treatment.[4-7] As with TMPT there might be an association with homozygous ITPA 94C-A patients and thiopurine toxicity, but thus far insufficient numbers of homozygous patients have been tested to allow a firm conclusion. Thus in our minds it is to early to conclude that the ITPA 94C>A polymorphism is associated with a higher risk at the development of side-effects on AZA treatment.

  References

  1. Marinaki AM, Ansari A, Duley JA, et al. Adverse drug reactions to azathioprine therapy are associated with polymorphism in the gene encoding inosine triphosphate pyrophosphatase (ITPase). Pharmacogenetics. 2004;14:181-187.

  2. von Ahsen N, Armstrong VW, Behrens C, et al. Association of Inosine Triphosphatase 94C>A and Thiopurine S-Methyltransferase Deficiency with Adverse Events and Study Drop-Outs under Azathioprine Therapy in a Prospective Crohn Disease Study. Clin Chem. 2005.

  3. Lander E, Kruglyak L. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet. 1995;11:241-247.

  4. Breen DP, Marinaki AM, Arenas M, et al. Pharmacogenetic association with adverse drug reactions to azathioprine immunosuppressive therapy following liver transplantation. Liver Transpl. 2005;11:826-833.

  5. Gearry RB, Roberts RL, Barclay ML, et al. Lack of association between the ITPA 94C>A polymorphism and adverse effects from azathioprine. Pharmacogenetics. 2004;14:779-781.

  6. Hindorf U, Lindqvist M, Strom M, et al. Lack of Association Between the Inosine Triphosphate Pyrophosphatase (ITPA) 94c>a Polymorphism, the Tpmt Genotype, the baseline Tpmt Activity and Adverse Effects From Thiopurine Medication in Inflammatory Bowel Disease. Digestive Disease Week. 2005.

  7. van Dieren JM, van Vuuren AJ, Kusters JG, et al. ITPA genotyping is not predictive for the development of side effects in AZA treated inflammatory bowel disease patients. Gut. 2005;54:1664.

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  Conflict of Interest:
  None declared.

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• Published on: 14 November 2005

  Is ITPA genotyping predictive for the development of side effects to AZA?

  • John A Duley, Senior Scientist
  • Other Contributors:
    • Anthony M. Marinaki, Monica Arenas, and Timothy H. J. Florin

  Dear Editor

  In a retrospective study of patients with inflammatory bowel disease, van Dieren et al recently reported the absence of correlation between genotypes for both inosine triphosphate pyrophosphatase (ITPA) thiopurine methyltransferase (TPMT) with any side effects to azathioprine (AZA) (Gut 2005;54:1664). This contrasts with two other studies. A rigorous prospective study recently published has demonstrat...

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  Dear Editor

  In a retrospective study of patients with inflammatory bowel disease, van Dieren et al recently reported the absence of correlation between genotypes for both inosine triphosphate pyrophosphatase (ITPA) thiopurine methyltransferase (TPMT) with any side effects to azathioprine (AZA) (Gut 2005;54:1664). This contrasts with two other studies. A rigorous prospective study recently published has demonstrated significant association between ITPA genotype and early drop-out from AZA therapy.(1) Our original publication implicated ITPA in a number of adverse effects, which were independent of myelosuppression.(2) One letter has reported non -association of ITPA specifically with myelosuppression,(3) but we have previously pointed out that thiopurine induced myelosuppression is well documented over the past 25 years to be associated with TPMT, not ITPA status.(4)

  However, we draw attention to a curious feature of the TPMT results of van Dieren et al, that one patient - who suffered severe myelosuppression - was reported as a TPMT*3B/*3B genotype. We previously published a meta-analysis of the incidence of the TPMT*3B mutant allele,(5) discovering that it is extremely rare, and this has been confirmed by a recent large study, making the chance of *3B homozygosity negligible.(6) Indeed, our evidence suggested that even the few cases of TPMT*3B may be over-reported, as a result of a technical problem in TPMT genotyping by PCR-RFLP. The A719G TPMT*3C mutation is usually identified by restriction endonuclease digestion that relies on creation of an Acc1 recognition site, and we have shown that this method is prone to failure.(5) The TPMT*3A allele, the most common mutant polymorphism among Caucasians, is a double mutant combining the TPMT*3C mutation and the G460A TPMT*3B mutation. A failure of A719G recognition will thus result in misreading the TPMT*3A allele as TPMT*3B (and TPMT*3C as wild-type TPMT*1).

  Curiously, van Dieren et al state that pre-therapy TPMT genotyping is “of limited clinical value” but their results show that the two cases of severe and potentially life threatening myelosuppression in their patient group were predicted by TPMT genotyping. In the absence of sequencing evidence, a cloud of doubt remains over the other TPMT genotype results presented. But until extensive and preferably prospective studies are accumulated in the literature it seems too early to dismiss the full pharmacogenetic value of ITPA and TPMT. In large scale clinical practices TPMT requesting is proving popular and useful, particularly in combination with thioguanine nucleotide monitoring.(6-8) We keenly await further publications elucidating genetic regulation of thiopurine metabolism as a valuable pharmacogenetic model.

  Conflict of interest

  None declared.

  References

  1. von Ahsen N, Armstrong VW, Behrens C, et al. Association of Inosine Triphosphatase 94C>A and Thiopurine S-Methyltransferase Deficiency with Adverse Events and Study Drop-Outs under Azathioprine Therapy in a Prospective Crohn Disease Study. Clin Chem 2005;51:2074-84

  2. Marinaki AM, Ansari A, Duley JA, et al. Adverse drug reactions to azathioprine therapy are associated with polymorphism in the gene encoding inosine triphosphate pyrophosphatase (ITPase). Pharmacogenetics 2004;14:181-7

  3. Allorge D, Hamdan R, Broly F, et al. ITPA genotyping test does not improve detection of Crohn's disease patients at risk of azathioprine/6- mercaptopurine induced myelosuppression. Gut 2005;54:565

  4. Sanderson J, Ansari A, Marinaki T, et al. Thiopurine methyltransferase: should it be measured before commencing thiopurine drug therapy? Annals of Clinical Biochemistry 2004;41:294-302

  5. Brouwer C, Marinaki AM, Lambooy LH, et al. Pitfalls in the determination of mutant alleles of the thiopurine methyltransferase gene. Leukemia 2001;15:1792-3

  6. Dervieux T, Meyer G, Barham R, et al. Liquid chromatography-tandem mass spectrometry analysis of erythrocyte thiopurine nucleotides and effect of thiopurine methyltransferase gene variants on these metabolites in patients receiving azathioprine/6-mercaptopurine therapy. Clin Chem 2005;51:2074-84.

  7. Gearry RB, Barclay ML, Roberts RL, et al. Thiopurine methyltransferase and 6-thioguanine nucleotide measurement: early experience of use in clinical practice. Intern Med J 2005;35:580-5.

  8. Holme SA, Duley JA, Sanderson J, et al. Erythrocyte thiopurine methyl transferase assessment prior to azathioprine use in the UK. Qjm 2002;95(7):439-44.

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  Conflict of Interest:
  None declared.

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