Frequent HPRT mutations in paroxysmal nocturnal haemoglobinuria reflect T cell clonal expansion, not genomic instability

Abstract

Summary 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 Vβ2 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.

The mutation rate in PIG-A is normal in patients with paroxysmal nocturnal hemoglobinuria (PNH)

“…The increased frequency of circulating HPRT mutants and glycophorin A variants in PNH had also been taken as evidence of hypermutability. 19,20,22,23 Chen et al 21 replicated such findings but provided data implicating increased cell divisions rather than hypermutability as the explanation. Our work now addresses the issue directly in 2 ways: (1) We use the same gene that is mutated in PNH as our sentinel gene, and (2) we eliminate preexisting mutants from the population to control for cell divisions and to calculate the mutation rate.…”

“…[15][16][17][18] Indeed, it is not uncommon for patients to harbor distinct red cell populations with a partial and complete lack of CD59 (termed PNH II and PNH III cells 4 ), which likely reflects the presence of distinct clones with partial and complete loss of GPI-anchor synthesis, respectively. 17 While the PIG-A mutant clones in patients are at least 4 orders of magnitude larger than in healthy individuals (eg, 1:5 versus 1:100 000), 5 smaller relative increases in the frequency (f) of lymphocytes with mutations in HPRT (Xq26-q27.2) [19][20][21] and red cells with variants in glycophorin A (4q28.2-q31.1) 22 have also been found in some patients with PNH. These findings have been regarded as suggestive of an increased mutation rate in PIG-A itself.…”

HPRT mutations, TCR gene rearrangements, and HTLV‐1 integration sites define in vivo T‐cell clonal lineages

“…Studies have now been reported for multiple sclerosis [Allegretta et al, 1990], Guillain-Barre syndrome [Van den Berg et al, 1995], systemic lupus erythematosus Wood et al, 1994;Theocharis et al, 1995], systemic sclerosis [Sfikakis et al, 1994], mixed connective tissue disease [Holyst et al, 1994], rheumatoid arthritis [Cannons et al, 1998], type 1 diabetes mellitus [Falta et al, 1999[Falta et al, , 2000, paroxysmal nocturnal hemoglobinuria [Chen et al, 2004], and malignant melanoma , with results consistent with the basic hypothesis [Albertini, 2001]. On a practical level, it has been found that sudden and dramatic elevations of HPRT MFs in cardiac transplant recipients have been reliable noninvasive indicators of rejection episodes [Ansari et al, 1995].…”

No evidence of hypermutability in red cells from patients with paroxysmal nocturnal hemoglobinuria using the XK gene

“…Others have reported an increased frequency of HPRT-mutants, 5,6 a possible consequence of increased lymphocyte turnover, rather than hypermutability. 7 We previously showed that GPI + cells from patients with PNH demonstrate no increase in the mutation rate in the PIG-A gene itself. 8 However, these prior studies, including our own, utilized lymphoid cells, whereas PNH is a stem cell disorder particularly affecting the myeloid/erythroid lineages.…”