Ann Lab Med 2019; 39(5): 438-446  https://doi.org/10.3343/alm.2019.39.5.438
Clonal Cell Proliferation in Paroxysmal Nocturnal Hemoglobinuria: Evaluation of PIGA Mutations and T-cell Receptor Clonality
Joonhong Park , M.D.1,2,*, Myungshin Kim , M.D.1,2,*, Yonggoo Kim , M.D., Ph.D.1,2, Kyungja Han , M.D.1,2, Nack-Gyun Chung , M.D.3, Bin Cho , M.D.3, Sung-Eun Lee , M.D.4, and Jong Wook Lee , M.D., Ph.D.4
1Department of Laboratory Medicine, 2Catholic Genetic Laboratory Center, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea; 3Department of Pediatrics, College of Medicine, The Catholic University of Korea, Seoul, Korea; 4Division of Hematology, Department of Internal Medicine, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea
Corresponding author: Yonggoo Kim, M.D., Ph.D.
Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea
Tel: +82-2-2258-1642, Fax: +82-2-2258-1719, E-mail: yonggoo@catholic.ac.kr
Jong Wook Lee, M.D., Ph.D.
Division of Hematology, Department of Internal Medicine, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea
Tel: +82-2-2258-6050, Fax: +82-2-780-1283, E-mail: jwlee@catholic.ac.kr
*These authors contributed equally to this study.
Received: March 5, 2018; Revised: August 23, 2018; Accepted: March 29, 2019; Published online: September 1, 2019.
© Korean Society for Laboratory Medicine. All rights reserved.

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Abstract
Background: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired pluripotent hematopoietic stem cell disorder associated with an increase in the number of glycosyl-phosphatidyl inositol (GPI)-deficient blood cells. We investigated PNH clonal proliferation in the three cell lineages—granulocytes, T lymphocytes, and red blood cells (RBCs)—by analyzing PIGA gene mutations and T-cell receptor (TCR) clonality.
Methods: Flow cytometry was used on peripheral blood samples from 24 PNH patients to measure the GPI-anchored protein (GPI-AP) deficient fraction in each blood cell lineage. PIGA gene mutations were analyzed in granulocytes and T lymphocytes by Sanger sequencing. A TCR clonality assay was performed in isolated GPI-AP deficient T lymphocytes.
Results: The GPI-AP deficient fraction among the three lineages was the highest in granulocytes, followed by RBCs and T lymphocytes. PIGA mutations were detected in both granulocytes and T lymphocytes of 19 patients (79.2%), with a higher mutation burden in granulocytes. The GPI-AP deficient fractions of granulocytes and T lymphocytes correlated moderately (rs=0.519, P=0.049) and strongly (rs=0.696, P=0.006) with PIGA mutation burden, respectively. PIGA mutations were more frequently observed in patients with clonal rearrangements in TCR genes (P=0.015). The PIGA mutation burden of T lymphocytes was higher in patients with clonal TCRB rearrangement.
Conclusions: PIGA mutations were present in approximately 80% of PNH patients. PNH clone size varies according to blood cell lineage, and clonal cells may obtain proliferation potential or gain a survival advantage over normal cells.
Keywords: Paroxysmal nocturnal hemoglobinuria, PIGA mutation, T-cell receptor clonality



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