Impaired growth and elevated Fas receptor expression in PIGA⁺ stem cells in primary paroxysmal nocturnal hemoglobinuria

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Abstract

The genetic defect underlying paroxysmal nocturnal hemoglobinuria (PNH) has been shown to reside in PIGA, a gene that encodes an element required for the first step in glycophosphatidylinositol anchor assembly. Why PIGA-mutated cells are able to expand in PNH marrow, however, is as yet unclear. To address this question, we compared the growth of affected CD59–CD34+ and unaffected CD59+CD34+ cells from patients with that of normal CD59+CD34+ cells in liquid culture. One hundred FACS-sorted cells were added per well into microtiter plates, and after 11 days at 37°C the progeny were counted and were analyzed for their differentiation pattern. We found that CD59–CD34+ cells from PNH patients proliferated to levels approaching those of normal cells, but that CD59+CD34+ cells from the patients gave rise to 20- to 140-fold fewer cells. Prior to sorting, the patients’ CD59– and CD59+CD34+ cells were equivalent with respect to early differentiation markers, and following culture, the CD45 differentiation patterns were identical to those of control CD34+ cells. Further analyses of the unsorted CD59+CD34+ population, however, showed elevated levels of Fas receptor. Addition of agonist anti-Fas mAb to cultures reduced the CD59+CD34+ cell yield by up to 78% but had a minimal effect on the CD59–CD34+ cells, whereas antagonist anti-Fas mAb enhanced the yield by up to 250%. These results suggest that expansion of PIGA-mutated cells in PNH marrow is due to a growth defect in nonmutated cells, and that greater susceptibility to apoptosis is one factor involved in the growth impairment.

Authors

Rui Chen, Shanmugam Nagarajan, Gregory M. Prince, Uma Maheshwari, Leon W.M.M. Terstappen, David R. Kaplan, Stanton L. Gerson, Jeffrey M. Albert, Daniel E. Dunn, Hillard M. Lazarus, M. Edward Medof