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

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 (r(s)=0.519, P=0.049) and strongly (r(s)=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.