Ann Lab Med.  2019 Sep;39(5):438-446. 10.3343/alm.2019.39.5.438.

Clonal Cell Proliferation in Paroxysmal Nocturnal Hemoglobinuria: Evaluation of PIGA Mutations and T-cell Receptor Clonality

• Park J
• Kim M
• Kim Y
• Han K
• Chung NG
• Cho B
• Lee SE
• Lee JW

• KMID: 2450966
• DOI: http://doi.org/10.3343/alm.2019.39.5.438

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.

Keyword

Paroxysmal nocturnal hemoglobinuria; PIGA mutation; T-cell receptor clonality

MeSH Terms

Blood Cells
Cell Proliferation*
Clone Cells
Erythrocytes
Flow Cytometry
Genes, T-Cell Receptor
Granulocytes
Hematopoietic Stem Cells
Hemoglobinuria, Paroxysmal*
Humans
Inositol
Receptors, Antigen, T-Cell*
T-Lymphocytes*
Inositol
Receptors, Antigen, T-Cell

Figure

• 

  Fig. 1 Spearman's correlations between GPI-AP deficient fractions of each cell lineage. (A) Correlation between GPI-AP-deficient fraction of RBCs (%) and GPI-AP-deficient fraction of granulocytes (%). (B) Correlation between GPI-AP deficient fraction of RBCs (%) and reticulocytes (%).Abbreviations: GPI-AP, glycosyl-phosphatidyl inositol-anchored protein; RBCs, red blood cells.

• 

  Fig. 2 Spearman's correlations between PIGA mutation burden and GPI-AP deficient fractions of the three cell lineages. (A) Correlation between GPI-AP deficient fraction of RBCs (red) and granulocytes (blue) (%) and mutation burden of granulocytes (%). (B) Correlation between GPI-AP deficient fraction of T lymphocytes (%) and mutation burden of T lymphocytes (%).Abbreviations: GPI-AP, glycosyl-phosphatidyl inositol-anchored protein; RBCs, red blood cells.

• 

  Fig. 3 Comparison of PIGA mutation burden and GPI-AP deficient fractions of T lymphocytes according to TCRB clonality. (A) PIGA mutation burden of T lymphocytes was significantly higher in patients with TCRB clonality than in patients without TCRB clonality (P=0.015). (B) The GPI-AP deficient fraction of T lymphocytes was higher, albeit not significantly, in patients with TCRB clonality than in patients without TCRB clonality (P=0.069).Abbreviation: GPI-AP, glycosyl-phosphatidyl inositol-anchored protein.

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