Thiopurines are an important therapy in the management of many autoimmune conditions (autoimmune hepatitis, inflammatory bowel disease [IBD], sarcoidosis, etc.), malignancies (lymphomas), and post-transplantation immunosuppression. The metabolism of thiopurines is complex, with numerous basic intermediates, such as nucleoside triphosphates, 6-thio-GTP, and 6-thio-dGTP. Polymorphisms that impact the activity of thiopurine methyltransferase (TPMT) could lead to leukopenia. NUDT15 gene polymorphisms have also been reported to be more important in East Asian and South Asian populations1,2. However, not all events of cytopenia are explained by the polymorphisms of those 2 genes, and many other genes have been proposed as possible reasons for thiopurine-related cytopenia.

The inosine triphosphate pyrophosphatase (ITPA) enzyme aids in the metabolism of azathioprine and 6-mercaptopurine. The information in the literature on the role of ITPA polymorphisms in causing thiopurine-related leukopenia is conflicting3–5. Initial reports suggested that ITPA mutations were associated with leukopenia, but those studies were primarily conducted in the Western world, including the Netherlands, Germany, the United Kingdom, and Venezuela3–5. However, a meta-analysis that included studies from Sweden, the Netherlands, and New Zealand showed no association between ITPA polymorphisms and side effects of thiopurines, such as myelosuppression5,6.

There is no available data regarding the relevance of ITPA polymorphisms in South Asian patients, including India. To address this knowledge gap, our aim was to study the impact of ITPA mutations on patients with IBD at a center in North India. We included the IBD patients that did not have the TPMT and NUDT15 polymorphisms, evaluating whether the presence of the ITPA (C.94C > A) polymorphism had any association with thiopurine-related leukopenia.

A total of 119 patients on thiopurines at our IBD clinic were initially selected. Leukopenia (WBC count < 3000/mm3) was seen in 33 (27.73%) patients. Of the 33 patients with leukopenia, 8 patients had the NUDT15 or TPMT polymorphisms, explaining the leukopenia. The TPMT mutation was seen in one patient with leukopenia, whereas the NUDT15 polymorphism was seen in 7. Therefore, 111 patients were included in the present study to assess the ITPA polymorphism (Fig. 1). The mean age of the patients was 36.36 ± 13.54 years, and 51.3% of the cases were males (Table 1). Regarding disease characteristics, 88.28% of the cases presented with ulcerative colitis and the remaining patients had Crohn’s disease. The median disease duration was 36 months, the median duration of thiopurine therapy was 8.5 months, and the median maximum tolerated dose was 100 mg.

Figure 1.

Flow of patients included in the study.

(0.13MB).

Of the 111 patients, 25 (21.01%) presented with unexplained leukopenia (Table 2). Four patients (16%) that developed leukopenia had the ITPA polymorphism (C.94C > A). Of the patients that did not develop leukopenia while taking thiopurines, 24 (27.9%) presented with the ITPA polymorphism. There was no significant association between the presence of leukopenia and the ITPA polymorphism (p = 0.228). Patients with IBD that had leukopenia were on lower tolerated azathioprine equivalent doses (p = 0.001) and also had a lower duration of thiopurine therapy (p = 0.015).

With respect to the other parameters, there was no significant association between sex (p = 0.326), IBD type (p = 0.143) or risk of idiosyncratic reaction (p = 0.18), and the adverse event of thiopurine-related leukopenia. The odds ratio for predicting leukopenia with the ITPA polymorphism was 0.4921 (95% CI 0.1520–1.5830, p = 0.234).

Thiopurines are used for the treatment of IBD because of their role as steroid-sparing agents in long-term maintenance therapy. Thiopurines are known to cause side effects, such as leukopenia, pancreatitis, hepatotoxicity, and gastrointestinal intolerance9. Those adverse events can lead to thiopurine discontinuation or disruption. The role of TPMT testing for thiopurine maintenance is well recognized. NUDT15 mutations are more frequently reported in the South Asian population than TPMT mutations and they are significantly associated with adverse events1. The FDA presently recommends testing for TPMT and NUDT15 in patients that develop severe myelotoxicity or repeated myelosuppression during thiopurine therapy. The role of ITPA in predicting myelosuppression in the South Asian population is largely unknown. In our prospective study on a South Asian population, the ITPA (C.94C > A) polymorphism was detected in 28 patients (25.2%) with IBD, making it a more common polymorphism than NUDT15 or TPMT. The ITPA polymorphism was detected in 16% of the patients that developed leukopenia, compared with 27.9% of the patients that did not develop leukopenia. We demonstrated that the ITPA polymorphism was not a predictor for leukopenia in patients on thiopurine therapy.

Inosine triphosphate pyrophosphatase helps convert inosine triphosphate (ITP) to inosine monophosphate (IMP). Patients on thiopurine with ITPA polymorphisms may have an increased accumulation of ITP. However, the clinical significance of those changes is unclear. The gene location for ITPA is chromosome 20. There are at least 5 single nucleotide polymorphisms reported for ITPA10. G138A, G561A, and G708A are usually silent, whereas the two remaining polymorphisms (C94A and IVS2 + A21C) are associated with reduced ITPA activity. The most commonly reported ITPA polymorphism is C94A. The mutations can be homozygous or heterozygous. ITPA deficiency leads to the accumulation of ITP nucleotides and toxic 6-thio-ITP. Theoretically, ITPA polymorphisms can cause cytopenia.

Asians (14-19%) harbor ITPA mutations more commonly than Whites and Africans (6-7%)11. A study of 63 patients with acute lymphoblastic leukemia from Northern India found an ITPA mutation in 17.5%12. Another report on 82 Lithuanian patients with IBD found no statistically significant impact of ITPA polymorphisms on leukopenia13. A study on 232 patients with IBD from Venezuela revealed a positive correlation between an ITPA polymorphism and azathioprine-related adverse events, such as myelosuppression and joint pain14. A Japanese study revealed that seven patients with IBD (from a total of 19 cases with an ITPA polymorphism) developed leukopenia15. A recent large Chinese study on 1419 patients with various diseases found no statistically significant association of the ITPA (94C > A) polymorphism with leukopenia16, but those authors recommended ITPA and NUDT15 genotype testing, before starting thiopurine therapy. They attributed various epigenetic factors associated with NUDT15 to the occurrence of adverse events. In our study, ITPA mutations were found in 25.2% of patients. We found no association between ITPA mutations and leukopenia. Therefore, testing for ITPA polymorphisms prior to the start of thiopurine therapy in Asian populations, such as ours, might not be clinically helpful in predicting leukopenia.

The strength of our study lies in the fact that it reports on the ITPA (C.94C > A) polymorphism in patients with IBD in South Asia. An important limitation of the study is its short follow-up of the patients on thiopurines. A long-term follow-up period is necessary for monitoring the side effects of thiopurines, given that leukopenia could also occur later in the disease course. Because azathioprine biologically converts to 6-MP and the dose relationship between the two is linear, we have included the single patient in whom 6-MP was used.

In addition, the polymorphism analyses are relevant to both drugs. To ensure that the inclusion of that patient did not impact the dose-related analysis, we used the equivalent azathioprine dose (Table 2) for such an analysis.

In conclusion, ITPA mutations were seen in 25.2% of the Indian population studied and there was no significant association between adverse events and the ITPA polymorphism. The presence of an ITPA polymorphism was not a predictor for leukopenia. We suggest there is no need for routine ITPA testing in Indian patients prior to starting thiopurine therapy.

All patients gave their informed consent to participate in this research. The work complies with the current regulations on bioethical research. This study was conducted after approval from the institutional ethics committee. The authors declare that this article does not contain personal information that can identify the patients.

No financial support was received in relation to this study/article.

The authors declare that there is no conflict of interest.