Genetic instability and the etiology of somatic PIG-A mutations in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematologic disorder charact erized by acquired PIG-A gene mutations that lead to defective bioassembly of glycosylphosphatidylinositol (GPI) anchors and the absence of GPI-linked surface proteins. As the etiology of these acquired PIG-A gene mutations i s unknown, we hypothesized that patients with PNH have overall genetic inst ability and acquire somatic mutations throughout their genome. We first ana lyzed microsatellite sequences and found equivalent size variation using DN A from GPI-negative granulocytes compared with the DNA of paired GPI-positi ve B cell lines or normal granulocytes. We next quantitated the frequency o f mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt(-) ) gene locus, and found 1 PNH patient with a large increase in hprf mutant frequency (256.7 x 10(-6) vs. 27.8 +/- 19.9 x 10(-6) for normal adults) tha t was confirmed on 1 independent blood samples. We also quantitated "illegi timate" VDJ genetic recombination events between the T cell receptor V gamm a and J beta gene loci, and found a second PNH patient with a large increas e (43.5 events per mu g of DNA vs. 1.3 +/- 0.8 events per mu g of DNA for n ormal adults), confirmed on 4 independent DNA samples. Both of these PNH pa tients are young females with no history of aplastic anemia. Our data show that PNH patients can have increased numbers of acquired somatic mutations in gene loci distinct from PIG-A. These data suggest that genetic instabili ty may be associated with the development of PIG-A mutations that lead to t he clinical picture of PNH. (C) 1999 Academic Press.