Abstract

Since the pivotal revelation of the PIGA gene mutations responsible for glycosylphosphatidylinositol (GPI) anchor deficiency over 20 years ago, molecular and clinical research into the evolution of Paroxysmal Nocturnal Hemoglobinuria (PNH) has significantly advanced the current understanding of the disease, expanding upon the foundational work by numerous investigators over the previous two centuries. The discovery of multiple PIGA mutations in normal individuals using the bacterial toxin aerolysin as well as with florescent activated cell sorting (FACS) clearly demonstrated that PIGA mutations are common in normal hematopoiesis. A strong association of PNH with Aplastic Anemia (AA) and the failure of PIGA clones to expand in animal models argued for the necessity of permissive conditions, largely understood to be immune mediated bone marrow failure. While GPI anchor deficiency may lead to escape of the PNH clone from autoimmunity, recent research has added to the body of knowledge by demonstrating that the PNH clone may acquire additional mutations in other genes that promote clonal expansion in the absence of competition from normal hematopoiesis as found in AA.

Sequencing studies of PIGA in PNH patients suggested that one, two, or at most three hematopoietic stem cells were sufficient to supply the necessary blood cells for survival. Furthermore, specific monoclonal antibodies combined with the fluorescently labeled inactive proaerolysin variant (FLAER) currently used to perform PNH diagnostic assays visualized the relatively frequent occurrence of both Type II and Type III PNH cells, suggesting the presence of two PIGA mutations in a significant number of patients. Using a combination of multiparameter FACS and a custom designed multiamplicon next generation sequencing (NGS) assay targeting PIGA, our results suggest that this may be an underestimate.

17 sequential patients with PNH (N=7, 3 Male, 4 Female) or AA/PNH (N=10, 5 Male, 5 Female) were enrolled in this study. A flow cytometry panel consisting of CD235a/CD59 for RBCs and FLAER/CD24/CD15/CD45 for granulocytes was used to assess PNH clone size and to sort WBCs into PNH positive and negative fractions. Mean RBC clone size was 31.6% (range 1.1-63.5%); mean WBC clone size was 53.2% (range 0.17-99.7%). Sort purity was confirmed at >98% in both fractions, DNA was extracted and subjected to analysis using an NGS assay and a stringent bioanalytic pipeline with an average depth of 18,000 reads.

At least one PIGA mutation was detected in the PNH positive fraction of every patient. A total of 68 PIGA mutations were observed, consisting of 31 nonsynonymous SNVs, 16 frameshift insertion/deletions, 12 stopgains, 7 splice site, and 2 nonframeshift deletions. 13/17 (76%) had more than one mutation, and 12/17 (70%) had 3 or more mutations (range 3-14). Analysis of variant allelic frequency (VAF) indicated that multiple clones with distinct PIGA mutations greater than 5% VAF of the PNH positive fraction were found in 9/17 (53%) patients with a median VAF of 11% (range 5-86%) and 5/9 demonstrating 3 mutations >5%. Repeat identical experiments from three patients were performed on samples obtained roughly one month apart with concordant results. Overall, our results suggest that a complex clonal hierarchy with multiple dominant and/or subdominant yet expanded clones is relatively common in PNH.

The clonal hierarchy in PNH patients can include up to 14 PNH clones with distinct frequencies and mutations. In addition, it is a widely held notion that PIGA mutations occur only in a hematopoietic stem cell, thus affecting all lineages, yet anomalous cases of PNH have been reported where the RBC PNH clone size is markedly higher than that of the granulocyte clone, and comparison between monocyte and granulocyte clone size is significantly different. We have identified two such cases in this cohort, with flow cytometry revealing RBC PNH clones of 18.63% and 35.61%, while granulocyte clones were 0.17% and 8.11%, and monocyte clones were 53.12% and 50.35%, respectively. Current experiments isolating and sequencing both sorted PNH positive cell fractions as well as hematopoietic precursors for PIGA and other commonly mutated genes found in hematologic malignancies are underway to confirm and elucidate the complex clonal hierarchy these results suggest.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.