Deep sequencing reveals stepwise mutation acquisition in paroxysmal nocturnal hemoglobinuria
Wenyi Shen, … , Jaroslaw P. Maciejewski, Hideki Makishima
Wenyi Shen, … , Jaroslaw P. Maciejewski, Hideki Makishima
Published October 1, 2014; First published September 17, 2014
Citation Information: J Clin Invest. 2014;124(10):4529-4538. https://doi.org/10.1172/JCI74747.
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Category: Research Article

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.

Authors

Wenyi Shen, Michael J. Clemente, Naoko Hosono, Kenichi Yoshida, Bartlomiej Przychodzen, Tetsuichi Yoshizato, Yuichi Shiraishi, Satoru Miyano, Seishi Ogawa, Jaroslaw P. Maciejewski, Hideki Makishima

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Figure 1

PIGA mutations can be either primary or secondary events.

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PIGA mutations can be either primary or secondary events. The primary ev...
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.