International Journal of Hematology

DOI: 10.1007/s12185-018-2475-6 Pages: 1-10

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

1. Tokyo Medical and Dental University (TMDU), Department of Pediatrics and Developmental Biology, Graduate School of Medical and Dental Sciences

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

3. Kazusa DNA Research Institute, Department of Technology Development

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

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

6. Japanese Red Cross Osaka Hospital, Department of Pediatrics

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

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

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

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

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

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

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

Correspondence to:
Hirokazu Kanegane
Tel: +81-3-5803-5244
Email: hkanegane.ped@tmd.ac.jp

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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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