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Increased DNA-incorporated thiopurine metabolite as a possible mechanism for leukocytopenia through cell apoptosis in inflammatory bowel disease patients with NUDT15 mutation

Abstract

Background and Aims

Polymorphisms in the nucleotide diphosphate-linked moiety X-type motif 15 (NUDT15) gene are associated with thiopurine-induced leukopenia in patients with inflammatory bowel disease (IBD). NUDT15-associated subcellular thiopurine metabolism has not been investigated in primary lymphocytes. We hypothesized that NUDT15 mutation increases DNA-incorporated deoxythioguanosine (dTG) and induces apoptosis in lymphocytes.

Methods

DNA-incorporated dTG in peripheral blood mononuclear cells (PBMCs) and 6-thioguanine nucleotides (6-TGN) in red blood cells were measured in patients with IBD undergoing thiopurine treatment. The association of a single nucleotide polymorphism for NUDT15 (rs116855232) with dTGPBMC was examined. The pro-apoptotic effect of DNA-incorporated dTG was examined ex vivo in association with NUDT15 genotypes by co-culturing patient-derived peripheral CD4+ T lymphocytes with 6-thioguanine (6-TG).

Results

dTGPBMC was significantly higher in NUDT15 variants than in non-variants. dTGPBMC, but not 6-TGNRBC, negatively correlated with peripheral lymphocyte counts (r =  – 0.31 and  – 0.12, p = 0.012 and 0.173, respectively). DNA-incorporated dTG significantly accumulated to a greater extent in lymphocytes from NUDT15 variants when co-cultured with 6-TG ex vivo than in those from non-variants and was associated with decreased proliferation and increased apoptosis.

Conclusion

Increased DNA-incorporated dTG may be responsible for thiopurine-induced leukocytopenia through cell apoptosis in IBD patients with NUDT15 mutation.

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Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Abbreviations

CD:

Crohn’s disease

FBS:

Fetal bovine serum

HBSS:

Hanks’ balanced salt solution

IBD:

Inflammatory bowel disease

PBMC:

Peripheral blood mononuclear cell

PBS:

Phosphate buffered saline

RBC:

Red blood cell

SNP:

Single nucleotide polymorphism

UC:

Ulcerative colitis

WBC:

White blood cell

WST:

Water-soluble tetrazolium

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Acknowledgements

We would like to extend our sincere gratitude and appreciation to Tadae Mori (Department of Gastroenterology, Kitasato University Kitasato Institute Hospital), Yuki Watanabe, Toyomi Ishibashi (Center for Advanced IBD Research and Treatment, Kitasato University Kitasato Institute Hospital) for helping us to collect clinical samples and data.

Funding

This work was supported in Japan by a grant from the Japan Foundation for Applied Enzymology, Japan Society for the Promotion of Science KAKENHI Grant Number JP19K17443, and a Grant-in-Aid for the Intractable Disease Project of the Ministry of Health, Labour and Welfare of Japan. In the UK, measurement of dTG incorporation was supported by Newcastle Healthcare Charity, the JGW Patterson Foundation, and a Confidence in Concept award from the Medical Research Council.

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Authors

Contributions

TK devised the conceptual ideas of the study. TT, TK, SK, AU, HK, SO, OT, CPFR, HT, RO, SS, MN, SAC, YT, and TH all made substantial contributions to all of the following: design of the study, acquisition of data, analysis and interpretation of data, revising the article critically for important intellectual content, and final approval of the version to be submitted. TT and TK also contributed to drafting the article. All authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Corresponding author

Correspondence to Taku Kobayashi.

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Conflicts of interest

T. Toyonaga receives research grants from Pfizer Inc. T. Kobayashi receives lecture fees from Abbvie Inc, Kyorin Pharmaceutical, Mitsubishi Tanabe, EA Pharma, Medtronic Co., Ltd, Janssen, Mochida Pharmaceutical, Takeda Pharmaceutical, Gilead Sciences, Nippon Kayaku, JIMRO, ZERIA Pharmaceutical, Astellas, Asahi Kasei Medical, Thermo Fisher Scientific, and Pfizer, advisory/consulting board fees from Abbvie Inc, Alfresa Pharma, Celltrion, Pfizer, Eli Lilly, Ferring Pharmaceuticals, Covidien, Janssen, Mochida Pharmaceutical, Takeda Pharmaceutical, Gilead Sciences, Nippon Kayaku, ZERIA Pharmaceutical, Thermo Fisher Scientific, and EA Pharma, and grants from EA Pharma, Thermo Fisher Scientific, Alfresa Pharma, and Nippon Kayaku. T. Hibi receives lecture fees from Mitsubishi-Tanabe Pharma, Kyorin Pharmaceutical, Abbvie GK, Janssen, JIMRO Co., Ltd, EA Pharma, Mochida Pharmaceutical, Takeda Pharmaceutical, Gilead Sciences, Celltrion, Nippon Kayaku, Kissei Pharmaceutical, Miyarisan Pharmaceutical, Zeria Pharmaceutical, and Ferring Pharmaceutical, advisory/consulting board fees from Abbvie GK,Takeda Pharmaceutical, Mitsubishi-Tanabe Pharma, JIMRO Co., Ltd, EA Pharma, Eli Lilly, Pfizer Japan Inc, Nichi-Iko Pharmaceutical, and Nippon Kayaku, and research grants from EA Pharma, Abbvie GK, JIMRO Co., Ltd, Zeria Pharmaceutical, Otsuka Pharmaceutical Co., Ltd. M. Nakano receives lecture/consultant fees from Covidien, Mochida Pharmaceutical, Takeda Pharmaceutical, Zeria Pharmaceutical, Kyorin Pharmaceutical and Nippon Kayaku, and research fundings from Mitsubishi Tanabe Pharma and Japanese foundation for research and promotion of endoscopy. S. Sagami has served as a speaker for AbbVie, Takeda Pharmaceutical, Zeria Pharmaceutical, served as a consultant for Nippon Kayaku, and an endowed chair from AbbVie, EA Pharma, JIMRO, Kyorin Pharmaceutical, Mochida Pharmaceutical, and Zeria Pharmaceutical, and has received research funding from Japan Foundation for Applied Enzymology.

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Toyonaga, T., Kobayashi, T., Kuronuma, S. et al. Increased DNA-incorporated thiopurine metabolite as a possible mechanism for leukocytopenia through cell apoptosis in inflammatory bowel disease patients with NUDT15 mutation. J Gastroenterol (2021). https://doi.org/10.1007/s00535-021-01820-0

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Keywords

  • Inflammatory bowel disease
  • Thiopurine
  • NUDT15