Deep sequencing reveals stepwise mutation acquisition in paroxysmal nocturnal hemoglobinuria

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease of hematopoietic stem cells that is associated with hemolysis, marrow failure, and thrombophilia. PNH has been considered a monogenic disease that results from somatic mutations in the gene encoding PIGA, which is required for biosynthesis of glycosylphosphatidylinisotol-anchored (GPI-anchored) proteins. The loss of certain GPI-anchored proteins is hypothesized to provide the mutant clone with an extrinsic growth advantage, but some features of PNH argue that there are intrinsic drivers of clonal expansion. Here, we performed whole-exome sequencing of paired PNH+ and PNH- fractions on samples taken from 12 patients as well as targeted deep sequencing of an additional 36 PNH patients. We identified additional somatic mutations that resulted in a complex hierarchical clonal architecture, similar to that observed in myeloid neoplasms. In addition to mutations in PIGA, mutations were found in genes known to be involved in myeloid neoplasm pathogenesis, including TET2, SUZ12, U2AF1, and JAK2. Clonal analysis indicated that these additional mutations arose either as a subclone within the PIGA-mutant population, or prior to PIGA mutation. Together, our data indicate that in addition to PIGA mutations, accessory genetic events are frequent in PNH, suggesting a stepwise clonal evolution derived from a singular stem cell clone.

Figure 4. Mutational summary and clonal architecture scenarios.

(A) Cohort frequency of 4 different scenarios for the clonal architecture encountered in PNH: ancestral PIGA mutation (B), secondary PIGA mutation (C), PIGA mutation as an isolated genetic event (D), and PIGA mutation leading to a PNH clone that independently coexists with an MDS clone (E).

Figure 3. PIGA mutation as the initial ancestral event in PNH8.

(A) A PIGA mutation together with 3 other somatic mutations (C11orf34, RBP3, and MUC7) were detected via WES at various allelic frequencies. All mutations were confined to the PNH fraction. Of note is that, in this case, the PIGA mutation was hemizygous, rendering the clone size equivalent to the allelic frequency. (B) Single-colony Sanger sequencing further confirmed the results obtained from deep sequencing and suggested that a PIGA mutation was the initial event. n = the number of colonies with the indicated mutations observed. Colonies that were not reproducible in independent experiments are not shown. (C) Proposed model of clonal architecture in this case based on VAFs and colony sequencing results.

Figure 2. PIGA mutations and longitudinal analysis.

(A) Distribution of PIGA missense, frameshift, nonsense, splice site, and microdeletions. (B) Proportion and absolute number of patients with 1, 2, or 3 mutations as well as microdeletions and those patients with the PNH phenotype in which a PIGA mutation was not detected. (C) Representative flow cytometric plot quantifying the number of type I (normal), type II (intermediate GPI anchor loss), and type III (complete GPI anchor loss) cells. (D) Longitudinal flow cytometry quantifying the wbc PNH clone size in patients with approximately 4 years or more of follow-up (n = 16). Each time point corresponds to approximately 1 year.

Figure 1. PIGA mutations can be either primary or secondary events.

The primary event (PNH5) is represented in A and B; the secondary event (PNH1) is represented in C–F. (A) Analysis of VAFs of the mutations identified in case PNH5 indicated that the KDM3B mutation was present at a lower frequency than the PIGA mutation, and both mutations were almost exclusively confined to the sorted PNH⁺ (CD59^–) fraction. (B) Single-colony sequencing results confirmed that the PIGA and KDM3B mutations were present in the same cell population. (C) Deep sequencing VAFs for PIGA-1 (G68E), PIGA-2 (splice site), and NTNG1 (P24S) mutations, all of which were primarily present in the PNH fraction in the PNH1 case. (D) Bacterial subcloning and Sanger sequencing results demonstrated that the PIGA mutations in this case were independent, suggesting the presence of 2 separate PNH clones. (E) Single-colony sequencing further confirmed that 2 independent PNH clones were present and also suggested that the PIGA splice site mutation appeared to be a secondary event preceded by a NTNG1 mutation. (F) The combination of deep sequencing data with single-colony sequencing allowed for a representation of the clonal architecture in PNH1.