THE ETIOLOGY OF SOMATIC MUTATIONS IN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)

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PNH is an acquired clonal stem cell disorder characterized by a somatic mutation in the PIG-A gene. Although the molecular mechanism of intravascular hemolysis is well understood, the etiology of PIG-A gene mutation and the mechanism of selective expansion of affected cells still remain unknown. Based on the presence of multiple PIG-A mutations, more than one PIG-A mutant clones in a single patient and a coexistence with MDS or leukemia clones in some patients, we hypothesize the genetic instability in the overeall genome in PNH.To test this, three distinct markers of genetic instability, microsatellite instability (MSI), chromosomal abnormality and mutation rate of the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene were analyzed in PNH patients.We first examined MSI in 20 patients and found rarity of MSI as reported (Eur J Haematol, 64 : 430-2, 2000).To asses the chromosomal instability, aneuploidy for chromosome 7 and 8 was analyzed in 13 patients using G-banding and fluorescence in situ hybridization (FISH). Five (38%) had monosomy 7(-7) but none had trisomy 8, suggesting not only that chromosome 7 is genetically unstable in PNH but also that -7 is a useful marker for monitoring the disease progression to MDS or leukemia.Using HPRT gene mutation assay combined with T cell colony formation, we examined the frequency of mutations in T cell colonies. Mutant colonies were found in 8 of 12 (67%) patients and 3 of 17 (l8%) age-matched health volunteers (p<0.02). The incidence of mutant colonies was extremely higher in the patients (mean 84, x10^<-6>) than in the healthy donors (mean 1.3, x10^<-6>). Thus, the HPRT gene mutate more frequently in patients with PNH than in healthy controls.Taken together, we concluded that in PNH patients, conditions exist that favor the occurrence of diverse somatic mutations in blood cells.