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.

Clonal hematopoiesis in acquired aplastic anemia

“…A similar mechanism seems to operate in leukemic relapse after haploidentical transplantation, in which relapsed leukemic cells are thought to escape alloimmunity by losing the mismatched haploid alleles via 6pUPD. 75,76 A conspicuous correlation between AA and the PNH-type cells, together with the frequent presence of multiple PIGA-mutated clones in 10% to 30% of AA/PNH patients, 15,18,77,78 indicates a strong selective advantage of PIGA-mutated cells that would be sufficient for clonal expansion of PNH-type cells within BM in AA patients, 79,80 although there is no direct experimental evidence supporting this hypothesis. Nevertheless, this does not necessarily exclude a possible role of secondary mutations in clonal selection of PIGA-mutated cells, which has long been discussed.…”

“…Variious plausible secondary driver alterations have been reported in occasional cases and implicated in the clonal outgrowth of PIGA-mutated clones, including those affecting HMGA2, NRAS, JAK2, TET2, U2AF1, and even BCR/ABL. 78,[81][82][83][84] However, thus far, no consistently recurrent mutational targets have been identified, even with WES, 78 precluding the indispensable role of these secondary mutations in the development of PNH.…”

Chromosomal Aberrations and HMGA2 Expression in Paroxysmal Nocturnal Hemoglobinuria

“…Therefore, somatic mutations of the PIGA gene remain the main mechanism of PIGA disruption. These mutations are located throughout the entire coding region, and most are frameshift mutations producing at most a nonfunctional PIGA protein [19,29].…”

Nowe spojrzenie na nocną napadową hemoglobinurię

“…Według wczesnych teorii komórki pozbawione kotwicy GPI unikają ataku immunologicznego, ponieważ obiektem tego ataku mogą być białka GPI-AP, ale nie znaleziono na to dowodu [34,35]. Zgodnie z drugą teorią istnieje dodatkowa, choć dotąd niezidentyfikowana, mutacja lub dysregulacja epigenetyczna, dzięki której komórki klonu PNH mają przewagę proliferacyjną [30,36]. W myśl trzeciej teorii do dominacji komórek macierzystych GPI(-) dochodzi dzięki ich oporności na apoptozę.…”