Incidential Findings of Mutations in the PIGA Gene Are Highly Specific for the Presence of PNH Clones

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic anemia associated with severe thrombophilia and characterized by complement-mediated lysis of erythrocytes lacking glycosylphosphatidylinositol (GPI)-anchored proteins. In the majority of cases, GPI deficiency is caused by somatic mutations in the PIGA gene. Presence of PNH clones is associated with acquired aplastic anemia (AA) and can be found in patients with myelodysplastic syndrome (MDS) or rarely other myeloid neoplasms (MN). Flow cytometric analysis for deficiency of GPI-anchored proteins on multiple cell lineages detects PNH clones, and PIGA mutational analysis is not mandatory to establish the diagnosis. In contrast, molecular genetic analysis of targeted gene panels is widely used in the diagnostic workup of MN. We hypothesized that the inclusion of PIGA into the myeloid gene panel could identify obscure cases with PNH clones irrespective of the initial clinical suspicion.

Aim: To assess the significance of incidental findings of mutations in PIGA in the diagnostic workup of MN.

Methods: 20,320 consecutive patients undergoing sequencing analysis for a confirmed or suspected MN were analyzed for the presence of mutations in the PIGA gene. Patients with confirmed PNH analyzed only for PIGA were not included, and cases with previously known PIGA mutations were excluded from further analysis. DNA was isolated from peripheral blood (PB) or bone marrow, and sequencing was performed on NovaSeq after Illumina DNA Prep for Enrichment library preparation (Illumina, San Diego, CA) and hybrid capture of a 41 gene panel including the complete coding sequence of PIGA (IDT Inc., Coralville, IA); data was analyzed with Pisces and Pindel (BaseSpace, Illumina). Flow cytometry was performed on granulocytes, monocytes, and erythrocytes in PB using antibodies against GPI-anchored proteins (CD14, CD24, CD55, and CD59), fluorescein-labeled proaerolysin (FLAER) staining, and Navios cytometers; analysis was done using Kaluza software (both Beckman Coulter, Miami, FL).

Results: PIGA mutations were newly identified in 67 patients (0.3%) undergoing targeted sequencing within the diagnostic workup of MN. 30 patients were excluded from further analysis as the gene panel had been requested for a MN associated with previously diagnosed PNH. From the remaining patients, PB for flow cytometry analysis could be obtained from 20 patients. Flow cytometry confirmed the presence of a PNH clone in 17 (85%) of these patients (median clone size: 41% for granulocytes, 54.5% for monocytes, and 12% for erythrocytes). In 3 patients (15%) with unexpected PIGA mutations, flow cytometry detected no PNH clone. The type of PIGA mutations differed significantly in those cases: Patients in whom a PNH clone was confirmed, showed protein-truncating frame-shift (41%) or nonsense (6%) mutations, splice site mutations (18%), or multiple mutations (35%) including at least one protein-truncating mutation at a median variant allele frequency (VAF) of 15.4% (range 2.0% to 50.1%). In contrast, patients without a PNH clone showed only singular missense mutations of PIGA with a VAF of 3.6% to 5.3% (Figure 1). Final diagnoses in patients with confirmed clones were sole PNH (n=9), or PNH clone associated with MDS (n=4), AA (n=3), and AML (n=1), and additional mutations in other genes were observed in 9 cases. While the initial clinical presentation included the differential diagnosis of PNH in some of the patients, flow cytometry was requested as a direct result of the PIGA mutation in 4 cases with an accompanying MN and in 3 patients without - the later showing a median latency of 6.5 years from the initial clinical presentation to the diagnosis.

Conclusions: The inclusion of PIGA into a standardized targeted sequencing panel for MN helps to identify patients with PNH clones irrespective of the initial clinical suspicion but is not sufficient to rule out PNH. Protein-truncating PIGA mutations are highly specific for PNH clones whereas singular missense mutations may not necessarily effect GPI biosynthesis. Our data indicate that the incidental finding of a PIGA mutation in sequencing analysis shall entail flow cytometry of GPI-anchored proteins in PB. The potential clinical sequelae and the availability of specific treatment options such as complement inhibitors warrant the thorough exclusion of PNH in the diagnostic workup of suspected MN.

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