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Comprehensive molecular diagnosis of Epstein–Barr virus-associated lymphoproliferative diseases using next-generation sequencing

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Abstract

Epstein–Barr virus (EBV) is associated with several life-threatening diseases, such as lymphoproliferative disease (LPD), particularly in immunocompromised hosts. Some categories of primary immunodeficiency diseases (PIDs) including X-linked lymphoproliferative syndrome (XLP), are characterized by susceptibility and vulnerability to EBV infection. The number of genetically defined PIDs is rapidly increasing, and clinical genetic testing plays an important role in establishing a definitive diagnosis. Whole-exome sequencing is performed for diagnosing rare genetic diseases, but is both expensive and time-consuming. Low-cost, high-throughput gene analysis systems are thus necessary. We developed a comprehensive molecular diagnostic method using a two-step tailed polymerase chain reaction (PCR) and a next-generation sequencing (NGS) platform to detect mutations in 23 candidate genes responsible for XLP or XLP-like diseases. Samples from 19 patients suspected of having EBV-associated LPD were used in this comprehensive molecular diagnosis. Causative gene mutations (involving PRF1 and SH2D1A) were detected in two of the 19 patients studied. This comprehensive diagnosis method effectively detected mutations in all coding exons of 23 genes with sufficient read numbers for each amplicon. This comprehensive molecular diagnostic method using PCR and NGS provides a rapid, accurate, low-cost diagnosis for patients with XLP or XLP-like diseases.

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Acknowledgements

We thank the patients and their parents as well as the doctors who provided the samples. This study was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Ministry of Health, Labour, and Welfare of Japan, and the Japan Blood Products Organization.

Author information

Author notes

  1. Shintaro Ono and Manabu Nakayama have contributed equally to this work.

Authors and Affiliations

  1. Department of Pediatrics and Developmental Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan

    Shintaro Ono, Hirokazu Kanegane, Akihiro Hoshino, Tsubasa Okano, Motoi Yamashita & Tomohiro Morio

  2. Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Japan

    Shintaro Ono & Osamu Ohara

  3. Department of Technology Development, Kazusa DNA Research Institute, Chiba, Japan

    Manabu Nakayama & Osamu Ohara

  4. Department of Lifetime Clinical Immunology, Tokyo Medical and Dental University (TMDU), Tokyo, Japan

    Akihiro Hoshino

  5. Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan

    Saeko Shimodera, Hirofumi Shibata, Takahiro Yasumi & Kazushi Izawa

  6. Department of Pediatrics, Japanese Red Cross Osaka Hospital, Osaka, Japan

    Hisanori Fujino

  7. Department of Hematology, Japanese Red Cross Kyoto Daiichi Hospital, Kyoto, Japan

    Takahiro Fujino

  8. Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University (TMDU), Tokyo, Japan

    Yuta Yunomae

  9. Department of Community Pediatrics, Perinatal and Maternal Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan

    Masatoshi Takagi & Kohsuke Imai

  10. Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Kejian Zhang

  11. Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Rebecca Marsh

  12. Study Center for Primary Immunodeficiencies, Necker-Enfants Malades Hospital, Assistance Publique Hôpitaux de Paris (APHP), Necker Medical School, Paris, France

    Capucine Picard

  13. Laboratory of Lymphocyte Activation and Susceptibility to EBV Infection, Institut National de la Santé et de la Recherche Médicale (INSERM) UMR 1163, Imagine Institut, Paris, France

    Capucine Picard & Sylvain Latour

Authors
  1. Shintaro Ono
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  2. Manabu Nakayama
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  3. Hirokazu Kanegane
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  17. Rebecca Marsh
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  18. Capucine Picard
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  20. Osamu Ohara
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  21. Tomohiro Morio
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Corresponding author

Correspondence to Hirokazu Kanegane.

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Cite this article

Ono, S., Nakayama, M., Kanegane, H. et al. Comprehensive molecular diagnosis of Epstein–Barr virus-associated lymphoproliferative diseases using next-generation sequencing. Int J Hematol 108, 319–328 (2018). https://doi.org/10.1007/s12185-018-2475-6

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  • DOI: https://doi.org/10.1007/s12185-018-2475-6

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