Session: 101. Red Cells and Erythropoiesis, Structure and Function, Metabolism, and Survival, Excluding Iron: Poster III
Hematology Disease Topics & Pathways:
Anemias, Diseases, Bone Marrow Failure, Biological Processes, PNH, genomics, pathogenesis
Among 295 diagnosed patients who had a PNH clone (>0.5% of PNH granulocytes), we identified 60 (median age 37 years, range 5-84) who fulfilled the criteria for hemolytic PNH (PNH clone size >20% and LDH>x2.5 ULN), tested before complement blocker therapy. Whole blood DNA was subjected to multi-amplicon deep NGS using primers covering all exons of PIGA gene.2 Accordingly, 13/60 (21%) were classified as having TII and 47/60 (79%) TIII PNH. Overall, median granulocyte clone size was 71.5% (21.5-99) with no differences between TII/TIII subtypes (p=0.872) as well as it was for baseline characteristics such as sex ratio, blood counts and previous history of aplastic anemia.
A total of 137 PIGA mutations were found, 27 in TII and 110 in TIII dominant patients. (Fig.1A-B) Overall, >1 and >2 mutations (clonal mosaicism) were found in 85% and 57% of patients, respectively. In cases negative by NGS of PIGA gene (9/60, 89% TIII PNH), no mutations were found even after sorting to purity of GPI(+) cells. Frameshift (39%, n=54) and missense (30%, n=41) mutations were most common, followed by splice site (16%, n=22), non-sense (11%, n=15) and non frameshift insertion/deletion (4%, n=5). Although PIGA mutations are spread throughout the entire coding region, TIII patients harbouring non-frameshift hits had more often truncating mutations mapping within the first 200bps across the PIGA domain. We demonstrated that missense PIGA hits were enriched in TII PNH, while frameshift deletions in TIII dominant patients (p=.0021 and .0201, respectively- Fig.1C). Moreover, when only dominant clone was analyzed, missense and frameshift mutations were strong predictors of PNH RBC phenotype, defining TII RBC (p=.0002) and TIII RBC patients (p=.02), respectively. (Fig.1D-E) When patients harbouring 1 unique somatic hit were studied (16/60, 27%), missense mutations accounted only for 19% of cases. Indeed, in an internal cohort of 89 AA patients with small PNH granulocytes clones (<20%) missense mutations accounted for 43% of cases indicating incomplete immune privilege.
PIGA missense and truncating mutations dictate the susceptibility of PNH RBCs to complement. We show that while missense mutations may still lead to a deficient but not totally absent GPI-AP synthesis, truncating mutations seem to be responsible for an entirely disruptive phenotype, causing the complete absence of GPI-APs on RBC membrane surface. Sequencing of other genes in the GPI synthetic pathway (AbstractID# 139335) may help to identify other somatic hits explaining PNH phenotype of cases lacking PIGA.
Disclosures: Patel: Alexion: Other: educational speaker, Speakers Bureau. Maciejewski: Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau.
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