The spectrum of PIG-A gene mutations in aplastic anemia/paroxysmal nocturnal hemoglobinuria (AA/PNH): a high incidence of multiple mutations and evidence of a mutational hot spot

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) may arise during long-term followup of aplastic anemia (AA), and many AA patients have minor glycosylphosphatidylinositol (GPI) anchor-deficient clones, even at presentation. PIG-A gene mutations in AA/PNH and hemolytic PNH are thought to be similar, but studies on AA/PNH have been limited to individual cases and a few small series. We have studied a large series of AA patients with a GPI anchor-deficient clone (AA/PNH), including patients with minor clones, to determine whether their pattern of PIG-A mutations was identical to the reported spectrum in hemolytic PNH. AA patients with GPI anchor-deficient clones were identified by flow cytometry and minor clones were enriched by immunomagnetic selection. A variety of methods was used to analyze PIG-A mutations, and 57 mutations were identified in 40 patients. The majority were similar to those commonly reported, but insertions in the range of 30 to 88 bp, due to tandem duplication of PIG-A sequences, and deletions of more than 10 bp were also seen. In 3 patients we identified identical 5-bp deletions by conventional methods. This prompted the design of mutation-specific polymerase chain reaction (PCR) primers, which were used to demonstrate the presence of the same mutation in an additional 12 patients, identifying this as a mutational hot spot in the PIG-A gene. Multiple PIG-A mutations have been reported in 10% to 20% of PNH patients. Our results suggest that the large majority of AA/PNH patients have multiple mutations. These data may suggest a process of hypermutation in the PIG-A gene in AA stem

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.…”

High frequency of several PIG-A mutations in patients with aplastic anemia and myelodysplastic syndrome

“…12,[15][16][17] The proportions of GPI-deficient cells in AA and MDS patients varied greatly (0.003-99%) and were usually lower than those in PNH patients. [5][6][7][8][9][10][11][12][13][14][15][16][17] In addition, PIG-A mutations have partially been reported in patients with AA, 11,18 AA/PNH, 3,19 and MDS. 15,17 However, it could not be ruled out that these AA and MDS patients analyzed for PIG-A gene abnormality are actually PNH, because the AA and MDS patients had 2.4-28.5% and 1.19-22% of GPI-negative erythrocytes, respectively.…”

Antibodies to glycosylphosphatidyl-inositol anchored proteins (GPI-AP) in antithymocyte and antilymphocyte globulin: possible role for the expansion of GPI-AP deficient cells in aplastic anemia

“…These observations and the fact that many AA patients have minor GPI-AP deficient cells at diagnosis or emergence after immunosuppressive therapy [8][9][10][11][12][13][26][27][28] was the reason for us to investigate the antibody composition of ATG to GPI-anchored proteins. Indeed we could demonstrate the presence of high concentrations of CD52 antibodies and, somewhat lower, CD48 antibodies in all ATG products.…”

“…This clinical interrelation is based on a pathophysiological link: we and others could demonstrate that cells with a deficiency of glycosylphosphatidyl-inositol anchored pro-teins (GPI-AP) are present at diagnosis or emerge after immunosuppressive therapy in a substantial proportion of AA patients [8][9][10][11][12]. Like in primary, classical PNH, the GPI-AP deficient population in AA is characterized by mutations of the PIG-A gene [13]. These findings support the hypothesis that in many patients with AA PIG-A mutated, GPI-deficient cells are present at a low level.…”