PIG-a Mutations in a Brazilian Cohort of Patients with Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a disorder due to an acquired loss-of-function mutation in the phosphatidylinositol glycan class A (PIG-A) gene. A large spectrum of acquired PIG-A mutations has been described, like insertions or deletions involving a single base or several bases, and single base substitution that are the most common. Usually there are more than one PIGA mutations and one clone is predominant. The clinical manifestations of PNH are intrinsically related to clonal expansion of hematopoietic stem cell deficient in GPI-anchored proteins. Some hypothesis failed to explain alone this clonal expansion. Here we try to identify and to correlate PIG-A gene mutations with clinical manifestations in a series of patients with PNH.

Methods: We analyzed 31 patients with classical PNH (n=23) or aplastic anemia and PNH clone (n=8). The sequencing of the PIG-A gene was performed using the Sanger technique. The electropherograms were aligned against the reference sequence of the PIG-A gene deposited in GenBank (Accession number NG_009786), and analyzed using the Geneious R10 software (Biomatters). After analysis, a search was performed in the Clinvar, dbSNP and HGMD databases to verify the pathogenicity of the mutations. For variants without description in the literature, a pathogenicity prediction analysis was performed using Mutation Taster, Polyphen 2 and Human Splicing Finder software.

Results: We found 29 different variants of the PIG-A gene in 27 patients: 23 were new mutations, with no previous description in the literature, 3 were previously described mutations, and 3 were single nucleotide polymorphism (SNP). There was great variation in the type and location of somatic mutations. Mutations were predominantly small deletions and simple base changes; 42% of the mutations were described as frameshift mutations and 31% missense mutations. We did not find any specific correlation between the clinical characteristics of hemolytic PNH patients and their mutations, due to the wide variety of mutations. According the pathogenicity prediction programs, the majority (22 of 29) of the variants found were classified as probably pathogenic. Among the 23 patients with hemolytic PNH, 19 patients had at least one mutation classified as pathogenic. In patients with subclinical PNH, only SNPs were found. Fifteen patients with hemolytic PNH had more than one concomitant mutation, most of which were probably pathogenic mutations associated with a polymorphism.

Conclusion: We described PIG-A mutations in a series of PNH patients in Brazil and observed no correlation exists between mutation types and clinical features in hemolytic patients. Among subclinical PNH patients, only SNPs were observed, probably because of small clone sizes.