thioguanine-anhydrous and Hemoglobinuria--Paroxysmal

Paroxysmal nocturnal haemoglobinuria (PNH) results from acquired mutations in the PIG-A gene of an haematopoietic stem cell, leading to defective biosynthesis of glycosylphosphatidylinositol (GPI) anchors and deficient expression of GPI-anchored proteins on the surface of the cell's progeny. Some laboratory and clinical findings have suggested genomic instability to be intrinsic in PNH; this possibility has been supported by mutation analysis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene abnormalities. However, the HPRT assay examines lymphocytes in peripheral blood (PB), and T cells may be related to the pathophysiology of PNH. We analysed the molecular and functional features of HPRT mutants in PB mononuclear cells from eleven PNH patients. CD8 T cells predominated in these samples; approximately half of the CD8 cells lacked GPI-anchored protein expression, while only a small proportion of CD4 cells appeared to derive from the PNH clone. The HPRT mutant frequency (Mf) in T lymphocytes from PNH patients was significantly higher than in healthy controls. The majority of the mutant T lymphocyte clones were of CD4 phenotype, and they had phenotypically normal GPI-anchored protein expression. In PNH patients, the majority of HPRT mutant clones were contained within the Vbeta2 T cell receptor (TCR) subfamily, which was oligoclonal by complementarity-determining region three (CDR3) size analysis. Our results are more consistent with detection of uniform populations of expanded T cell clones, which presumably acquired HPRT mutations during antigen-driven cell proliferation, and not due to an increased Mf in PNH. HPRT mutant analysis does not support underlying genomic instability in PNH.

Topics: Adult; Cells, Cultured; Colony-Forming Units Assay; Complementarity Determining Regions; Female; Genomic Instability; Glycosylphosphatidylinositols; Hemoglobinuria, Paroxysmal; Humans; Hypoxanthine Phosphoribosyltransferase; Immunophenotyping; Male; Middle Aged; Mutation; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocyte Subsets; Thioguanine

Acquired mutations of the PIG-A gene result in the hemolysis characteristic of paroxysmal nocturnal hemoglobinuria (PNH). Although the etiology of the mutation(s) is unclear, mutable conditions have been suggested by the coexistence of multiple clones with different mutations of PIG-A and by the appearance of leukemic clones in patients with PNH. This study sought to test this hypothesis by examining the frequency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutations, identified by both resistance to 6-thioguanine (6-TG) and gene analysis. T-cell colonies resistant to 6-TG formed in methylcellulose culture were found in 8 (67%) of 12 PNH patients and 3 (18%) of 17 age-matched healthy volunteers (P <.02, Fisher exact probability test). The incidence of resistant colonies ranged from 40 to 367 (mean 149, x 10(-7)) in the 8 patients and from 1 to 16 (mean 7, x 10(-7)) in the 3 healthy donors. Thus, the HRPT gene mutated more frequently in patients with PNH than in healthy controls (P <.02, Mann-Whitney test). Analysis of bone marrow cells supported these findings. Like the PIG-A mutations in PNH, the HPRT mutations were widely distributed in the coding regions and consisted primarily of base deletions. Unlike PNH cells, 6-TG-resistant cells expressed CD59, indicating that the HPRT mutations did not occur in PNH clones. No correlation was noted between HPRT mutation frequency and content of therapy received by the patients. It is concluded that in PNH patients, conditions exist that favor the occurrence of diverse somatic mutations in blood cells.

Topics: Adult; Aged; Colony-Forming Units Assay; DNA Mutational Analysis; Drug Resistance; Female; Flow Cytometry; Glycosylphosphatidylinositols; Hemoglobinuria, Paroxysmal; Humans; Hypoxanthine Phosphoribosyltransferase; Male; Middle Aged; Mutation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes; Thioguanine