Endothelial Progenitor Cells in Paroxysmal Nocturnal Hemoglobinuria
Authors: Gandrille Sophie , de Latour Régis Peffault , Levionnois Emeline , Petropoulou Anna D. , Galy-Fauroux Isabelle , Rodriguez-Otero Paula , Socié Gérard , Fischer Anne-Marie , Helley Dominique 
Published: October 2019

Abstract 2397

Introduction:

Paroxysmal nocturnal hemoglobinuria (PNH), a very rare disease, is characterized by hemolytic anemia, bone marrow failure, and venous thromboembolism. The disease is caused by somatic mutation of the X-linked gene PIG-A encoding a key enzyme responsible for the biosynthesis of the GPI-anchored proteins (GPI-APs), affecting hematopoietic stem cells (HSC). Venous thromboembolisms occur in unusual sites and the mechanism remains to be elucidated. A previous study showed an increase of endothelial cell activation markers in PNH patients (Helley et al., Haematologica 2010;95:574-581), however endothelial cells remain poorly studied in this disease. In polycythemia vera, associating JAK-2 mutation in HSC and thrombosis occurrence, Sozer and coworkers showed that liver endothelial cells bore the same JAK-2 mutation than the HSC (Sozer et al, Blood Cells, Molecules and Diseases, 2009;43:304-312). This suggests the existence of a common precursor between endothelial progenitor cells and HSC, the hemangioblast.

In PNH, PIG-A gene mutations might be present in endothelial cells. To test this hypothesis we have used endothelial progenitor cells circulating in blood, also called endothelial-colony forming cells (ECFC), as witnesses of endothelial cells status.

Methods:

Peripheral blood mononuclear cells (PBMC) and neutrophiles (PMNL), from patients with classical PNH, were fractionated. PMNL were used to isolate DNA, while PBMC were plated on gelatine-coated plates to be cultured until appearance of ECFC colonies (usually 15–20 days), and then cultured to perform: (i) expression of GPI-anchored protein by flow cytometry; (ii) sequencing of the exons and flanking regions of the PIG-A gene from PMNL and ECFC.

Results:

Twelve PNH patients were enrolled (8 women, 4 men). Among them, 3 have venous thrombotic events (2 Budd-Chiari syndromes and one mesenteric veins thrombosis). ECFC colonies were obtained from 6 out of the 12 patients, a normal output due to the small number of ECFC in whole blood. Mutations identified by sequencing the PIG-A gene of PMNLs from these 6 patients are shown in Table 1, as well as clinical characteristics.

In 5 patients only (two with previous Budd-Chiari syndrome), we obtained sufficient amounts of ECFC to isolate DNA. None of the ECFC colonies bore the identified mutations (one exhibited a very faint peak of mutated nucleotide on electrophoregram but mRNA transcripts analysis from this ECFC colony revealed traces amounts of CD45 and CD11b mRNA, suggesting a contamination by leukocytes).

Conclusion:

This is the first study reporting the absence of PIG-A gene mutation in ECFC from PNH patients, indicating that the mature endothelial cells are not bearing the PIG-A mutation. These cells are thus involved in thrombosis probably by free hemoglobin and thrombin cell activation, rather than by direct damage caused by PIG-A gene mutation. These results also indicate that, in PNH, the PIG-A gene mutation occurs most probably after the common precursor between endothelial progenitor cells and HSC.

Disclosures:

Peffault de Latour: Alexion: Consultancy, Research Funding. Fischer:Alexion: Consultancy. Helley:Alexion: Consultancy.