Deep sequencing reveals stepwise mutation acquisition in paroxysmal nocturnal hemoglobinuria
Wenyi Shen, Michael J. Clemente, Naoko Hosono, Kenichi Yoshida, Bartlomiej Przychodzen, Tetsuichi Yoshizato, Yuichi Shiraishi, Satoru Miyano, Seishi Ogawa, Jaroslaw P. Maciejewski, Hideki Makishima
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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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 3

PIGA mutation as the initial ancestral event in PNH8.

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PIGA mutation as the initial ancestral event in PNH8. (A) A PIGA mutatio...
(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.