Exploring the Endothelial Cell Origins in PNH Patients

Erica Jones, Martin Rogers, Dr. Monica Bessler, Dept. of Hematology, Washington University School of Medicine

PNH (Paroxysmal Nocturnal Hemoglobinuria) patients form blood cells that lack the GPI anchor, which protects cells from immune responses. As a result, the PNH affected red blood cells are lysed and hemoglobin is released into the blood stream and expelled through the urine causing hemoglobinuria, thus, the name of the disease. PNH is an acquired disease and is caused by a somatic mutation in the PIGA gene occurring in a hematopoietic stem cell of the bone marrow. Therefore, PNH patients have a mixture of normal and PNH blood cells. Blood clotting is the leading cause of death in PNH patients but how this occurs is unknown. One possible reason is that not only the blood cells but also the endothelial cells are abnormal and lack GPI-linked proteins.

To test this hypothesis, blood was collected from a PNH patient and endothelial cell colonies were grown in an in vitro culture system. DNA was extracted from both the endothelial and blood cells. First, we screened the DNA from the blood cells for a mutation in the PIGA gene. The PIGA gene is located on the X chromosome and consists of 6 exons. We identified a small 45 bp deletion in exon 6 causing a 15 amino acid deletion in the PIGA protein. The deletion also causes a loss of a Fok I restriction enzyme site in exon 6. Next, we determined through PCR and subsequent digestion of the amplification product with Fok I whether or not the mutation was present in the patient's DNA isolated from total blood, granulocytes, mononuclear cells, bone marrow fibroblasts, and in DNA isolated from 4 independent endothelial cell colonies. We found that the mutation was present in DNA from total blood, granulocytes, mononuclear cells, but not in DNA obtained from bone marrow fibroblast or in DNA isolated form the endothelial cell colonies.

My results show that none of the four endothelial progenitor cells that gave rise to endothelial cell colonies in vitro carry the same abnormality as the blood cells from the same patient. This suggests that the PIGA gene mutation occurs in a progenitor cell that may give rise to all three blood cell lineages but not to endothelial cells. Although during embryogenesis a common progenitor exists that gives rise to both blood cells and endothelial cells it is highly controversial whether such a progenitor exists in the adult bone marrow. If such a progenitor exists analysis of additional patients and more colonies might be necessary.