Ultradeep Sequencing Analysis of Paroxysmal Nocturnal Hemoglobinuria Clones Detected by Flow Cytometry: PIG Mutation in Small PNH Clones.

Objectives: We aimed to determine whether small paroxysmal nocturnal hemoglobinuria (PNH) clones detected by flow cytometry (FCM) harbor PIG gene mutations with quantitative correlation.

Methods: We analyzed 89 specimens from 63 patients whose PNH clone size was ≥0.1% by FCM. We performed ultradeep sequencing for the PIGA, PIGM, PIGT, and PIGX genes in these specimens.

Results: A strong positive correlation between PNH clone size by FCM and variant allele frequency (VAF) of PIG gene mutation was identified (RBCs: r = 0.77, P < .001; granulocytes: r = 0.68, P < .001). Granulocyte clone size of 2.5% or greater and RBCs 0.4% or greater by FCM always harbored PIG gene mutations. Meanwhile, in patients with clone sizes of less than 2.5% in granulocytes or less than 0.4% in RBCs, PIG gene mutations were present in only 15.9% and 12.2% of cases, respectively. In addition, there was not a statistically significant positive correlation between FCM clone size and VAF or the presence or absence of a PIG mutation.

Conclusions: Our results showed that in small PNH clones PIG gene mutations were present in only a small portion without significant correlation to VAF or the presence or absence of a PIG mutation.