Ineffective erythropoiesis in recipient precursor cells following non-myeloablative stem cell transplantation in patients with severe sickle cell anemia

After nonmyeloablative (NMA) stem cell transplantation (SCT), we observed greater clinical improvement in sickle cell disease (SCD)-associated symptoms than anticipated by the degree of chimerism established by routine genotyping of marrow or peripheral blood cells in some patients. We hypothesized that RBC chimerism would provide a more accurate assessment of functional donor hematopoiesis. β-globin RNA pyrosequencing can be used to quantify erythroid-specific lineage chimerism following transplant. In this method, direct quantitative sequencing of the sickle mutation in β-globin RNA serves as an informative locus to distinguish between recipient and donor erythropoiesis. This analysis was performed on serial samples collected from 3 patients with severe SCD with HLA-matched related donors who underwent NMA-SCT. β-globin RNA chimerism in peripheral blood (PB) and marrow (BM) was compared to total genomic DNA chimerism and DNA chimerism of phenotypically defined erythroid lineage precursor cells. All patients demonstrated stable mixed donor DNA chimerism in the first 60–100 days, at levels of 25–30%, 40–50% and 56–60% in Pts 1, 2 and 3, respectively, with no difference between PB and BM levels. DNA chimerism of BM erythroid precursors, determined by Y chromosome FISH and ABO staining in Pts 1 and 2, were identical to total BM gDNA chimerism. In contrast, all 3 patients demonstrated a ∼2 fold greater level of β-globin RNA chimerism in PB and marrow compared with gDNA chimerism, with 45/55%, 100/100% and 100/100% BM/PB% donor β-globin RNA measured in Pts 1, 2 and 3, respectively. The consistently increased levels of donor derived β-globin RNA detected in marrow samples, compared to the lower levels of erythroid DNA chimerism suggest that recipient erythroid precursor cells with sickle hemoglobin do not effectively mature into peripheral RBC. In patients with mixed hematopoietic chimerism after NMA-SCT, donor erythroid precursor cells have a maturation advantage that allows for greater donor contribution to overall erythropoiesis. This advantage is likely further accentuated by the shortened survival of recipient RBC. Further studies are underway to characterize the mechanisms that lead to ineffective SCD erythropoiesis in these patients.