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Cytogenetics and molecular genetics

Detection of subclonal SETBP1 and JAK3 mutations in juvenile myelomonocytic leukemia using droplet digital PCR

Leukemia volume 35, pages 259–263 (2021)Cite this article

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Juvenile myelomonocytic leukemia (JMML) is an intractable and rare pediatric myelodysplastic/myeloproliferative neoplasm characterized by excessive myelomonocytic cell proliferation and granulocyte-macrophage colony-stimulating factor hypersensitivity [1]. More than 85% of patients with JMML harbor somatic and/or germline mutations in several RAS pathway genes such as PTPN11, NF1, NRAS, KRAS, and CBL [2,3,4]. This disease is genetically characterized by a small number of somatic mutations, except for the primary RAS pathway genes [5,6,7]. In a subset of patients with JMML, the secondary genetic events, including SETBP1 and JAK3 mutations, occur in addition to the primary RAS pathway mutations, and these mutations are associated with poor outcomes [5, 8]. Among the secondary mutations, most frequent hotspot mutations are SETBP1 p.D868N and JAK3 p.R657Q [9]. In the previous study, droplet digital PCR (ddPCR) identified subclonal SETBP1 hotspot mutations, including those with <0.1% variant allele frequency (VAF), among patients with JMML at initial diagnosis [10]. These rare SETBP1 mutations were independently correlated with a dismal prognosis. Similarly, we evaluated the subclonal JAK3 hotspot mutations with low VAF at a high sensitivity to reveal their clinical impact in JMML, and to investigate the correlations with subclonal SETBP1 hotspot mutations. Here, we assessed subclonal SETBP1 p.D868N and JAK3 p.R657Q hotspot mutations in patients with JMML and JMML-like myeloproliferative disorder associated with Noonan syndrome (NS/MPD) using ddPCR.

In this study, we enrolled 128 patients with JMML and 15 with NS/MPD (Supplementary Table 1); the same patients had been included in our previous publication [9]. ddPCR was performed to detect subclonal SETBP1 c.2602G>A (p.D868N) and JAK3 c.1970G>A (p.R657Q) hotspot mutations using the ddPCRTM supermix and a Bio-Rad QX200TM ddPCR analyzer (Bio-Rad, CA, USA; Supplementary Methods and Supplementary Table 2). This study was approved by the ethics committee of the Nagoya University Graduate School of Medicine and was conducted in accordance with the principles of the Declaration of Helsinki.

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Fig. 1: SETBP1 and JAK3 mutations identified by droplet digital PCR.
Fig. 2: Co-occurrence of SETBP1 and JAK3 mutations in JMML.

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Acknowledgements

The authors acknowledge all clinicians, patients, and their families, and thank Ms Yoshie Miura, Ms Chie Moriyama, Ms Hiroko Ono, and Ms Chie Amahori for their valuable assistance. The authors acknowledge the Division for Medical Research Engineering, Nagoya University Graduate School of Medicine, for providing technical support.

Funding

This work was supported by Japan Society for the Promotion of Science KAKENHI Grant number 18K07816 to HM.

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Authors and Affiliations

  1. Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan

    Manabu Wakamatsu, Norihiro Murakami, Shunsuke Miwata, Hironobu Kitazawa, Kotaro Narita, Shinsuke Kataoka, Daisuke Ichikawa, Motoharu Hamada, Rieko Taniguchi, Kyogo Suzuki, Nozomu Kawashima, Eri Nishikawa, Atsushi Narita, Nobuhiro Nishio, Seiji Kojima, Hideki Muramatsu & Yoshiyuki Takahashi

  2. Medical Genomics Center, Nagoya University Hospital, Nagoya, Japan

    Yusuke Okuno

  3. Center for Advanced Medicine and Clinical Research, Nagoya University Hospital, Nagoya, Japan

    Nobuhiro Nishio

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  1. Manabu Wakamatsu
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Contributions

MW performed research, analyzed data, and wrote the paper. YO, HM, and YT designed and analyzed data, led the project, and wrote the paper. NM, NK, and SK performed research and bioinformatics analysis. SM, KN, SK, DI, MH, RT, KS, NK, EN, AN, and NN collected clinical sample and data. All authors reviewed and approved the final version of the manuscript.

Corresponding authors

Correspondence to Hideki Muramatsu or Yoshiyuki Takahashi.

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The authors declare that they have no conflict of interest.

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Wakamatsu, M., Okuno, Y., Murakami, N. et al. Detection of subclonal SETBP1 and JAK3 mutations in juvenile myelomonocytic leukemia using droplet digital PCR. Leukemia 35, 259–263 (2021). https://doi.org/10.1038/s41375-020-0817-x

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