Abstract

The monitoring of lineage-specific engraftment is critical to understanding the impact of novel transplant regimens and determining how these can be modified to improve outcomes. We developed an RNA-based pyrosequencing assay to rapidly measure lineage-specific chimerism by quantification of cell type specific host versus donor transcripts that differ in the expression of single nucleotide polymorphisms (SNPs). To assess RBC lineage engraftment, we identified 10 common SNPs, expressed by 7 genes that encode RBC specific antigens and structural proteins by using the HapMap and Ensembl databases and direct high-throughput genotyping. These SNPs could then be PCR-amplified from total RNA extracted from peripheral blood, which contains nucleated erythroid progenitors. Mixing studies using samples of peripheral blood with defined alleles were performed to validate that each SNP could quantitatively measure donor/recipient DNA and RNA. Using this panel, we directly genotyped 15 patients and their HLA-matched related donors who underwent allogeneic hematopoietic stem cell transplantation for sickle cell disease (SCD) or thalassemia major. A median of 3 SNPs was informative for each donor/recipient pair. By using informative expressed RBC SNPs to quantify donor-derived RBC transcripts, we measured serial rates of erythroid lineage specific engraftment in 13 of the 15 patients that were compared to overall levels of donor mononuclear cell (WBC) engraftment. In pairs with greater than 1 informative SNP, high concordance in serial post-transplant chimerism measurements among individual SNPs was observed. At post-transplant day 30, 4 of 13 patients converted to full donor hematopoiesis, 1 demonstrated primary graft failure, and 8 developed partial donor WBC engraftment, ranging from 29 – 82%. Consistent with known ineffective erythropoiesis associated with SCD and thalassemia, we detected up to 3-fold greater RBC specific compared to overall WBC engraftment in 5 of 8 patients. In contrast, the remaining 3 of 8, all of whom received ABO-incompatible grafts, demonstrated at least 0.5-fold lower RBC compared to WBC engraftment. Detection of the effects of ABO incompatibility by RNA pyrosequencing was related to persistence of anti-isohemaglutinin antibodies. Since erythroid progenitors, the cell population evaluated by our assay, transit rapidly in peripheral blood relative to long-lived mature erythrocytes, RBC engraftment is potentially a sensitive marker for graft rejection. In keeping with this, 3 of 8 patients eventually rejected their grafts at 60, 219, and 288 days post-transplant, in which loss of WBC and RBC engraftment was concurrently detected. In summary, RNA pyrosequencing provides rapid measurement of erythroid lineage chimerism, without requiring specific cell isolation, and can provide valuable functional information for diseases in which RBC engraftment is critically important. Similar methods can be applied to generate panels of expressed SNPs informative for other cell lineages to assess the impact of novel stem cell therapies on lineage-specific engraftment.

Author notes

Disclosure: No relevant conflicts of interest to declare.