Detection of Myeloid and Clonal Expansion Related Gene Mutations By Whole-Exome Sequencing in Patients with Paroxysmal Nocturnal Hemoglobinuria

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a disease presented with hemolysis, cytopenia and thrombosis. Apart from PIGA gene on hematopoietic stem cells which accounts for the glycosylphosphatidylinositol (GPI) anchor deficiency on the cell membrane, other mutations have also been detected in PNH through whole-exome sequencing (WES). However, the characteristics of mutations in patients with PNH and genes which may contribute to PNH clonal expansion have not been well defined.

Methods: Peripheral blood samples were collected from 41 patients with PNH, among them samples from 6 patients were further separated into CD59- and CD59+ fractions by CD59 magnetic beads. Gene mutations were tested by whole-exome sequencing(WES). 178 genes commonly mutated in myeloid cancer were analyzed in the sequencing results, as well as their correlation with clinical indicators. Mutated genes correlated with cell proliferation were compared between sorted CD59+ and CD59- cells.

Results: The most frequently mutated myeloid cancer-related genes were MAP3K4 and CSMD1 (12.2% respectively). Among them, RUNX1T1 mutation was found to be correlated with larger clone size, higher level of uncombined bilirubin, and lower level of hemoglobin (P<0.05). No other correlation between clinical parameters and gene mutations were found. The proportion of mutations (DNMT3A、RUNX1、JAK2、JAK3、CSMD1) which have been shown to indicate poor outcome in patients with aplastic anemia decreased as PNH clone increased (p=0.026). Mutations related to cell proliferation tended to happen more frequently in CD59- fractions compared with CD59+ fractions of the same patient (P=0.062).

Conclusions: Myeloid cancer-related mutations can be detected in patients with PNH with some correlation with clinical manifestations. Larger PNH clone may "save" patients from mutation indicating poor prognosis. CD59- fractions seemed to carry more proliferation related mutations, which may contribute to PNH clonal expansion.