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

There are two models to account for the large population of GPI-negative cells and the occasional demonstration of oligoclonality in paroxysmal nocturnal hemoglobinuria (PNH): i) immune escape; and ii) hypermutability. In support of immune escape, in PNH, marrow failure is common and is associated with HLA-DR alleles and oligoclonal T-cell expansions. Conversely, in support of hypermutability, there is an increased relative risk of leukemia, and cytogenetic abnormalities and secondary mutations can occur. Others have reported an increased frequency of HPRT-mutants, a possible consequence of increased lymphocyte turnover, rather than hypermutability. We previously showed that GPI cells from patients with PNH demonstrate no increase in the mutation rate in the PIG-A gene itself. However, these prior studies, including our own, utilized lymphoid cells, whereas PNH is a stem cell disorder particularly affecting the myeloid/erythroid lineages. Here we have applied our analysis for spontaneously arising phenotypic variants using XK, the gene mutated in the McLeod syndrome, to explore this question. This assay has several advantages: 1) XK is X-linked, as are PIGA and HPRT, and thus a single mutation can produce the mutant phenotype; 2) a broad spectrum of mutations can inactivate the gene; 3) the XK mutant (McLeod) phenotype results in a loss of Kell proteins on red blood cells (RBC), which is detected by flow cytometry. Patients and normal donors provided written informed consent. Approximately 10 RBC from whole blood were incubated with 50 μL of MIMA91 supernatant (generated as described by Tearina et al.) which recognizes a non-polymorphic human Kell antigen. The RBC were washed twice with cold HANKS with 0.1% BSA and incubated with Rphycoerythrin-conjugated F(ab’)2 fragment rabbit antimouse immunoglobulin (Dako,1:5), washed twice, and incubated with anti-glycophorin-A-FITC (Dako, 1:10). We also analyzed thawed RBC from a patient with the McLeod syndrome and an obligate female carrier. Incubations were performed on ice for 30 min. Prior to each incubation, the cells were resuspended, pelleted, and resuspended. Some experiments used biotinylated antiCD59 (Serotec,1:20) and streptavidin-PerCP-Cy5.5 (Becton-Dickinson,1:2.5). Cells were analyzed on a BD FACScan using Cellquest and Flow-Jo. Voltage was adjusted so that unstained RBC had a mean fluorescence of 2.5 on FL1 and FL2; RBC were gated by FSC/SSC (log-log scale) and glycophorin-A expression. Cells having less than 20% of the mean FL2 of the overall population were defined as McLeod-like. First, MIMA91 was validated using RBC derived from a patient with the McLeod syndrome, an obligate carrier, and a normal donor (Figure 1A-C), and the patterns were similar to our previous data using an anti-K14 antibody. In the normal donor, there is a distinct population of RBC with the McLeod-like phenotype at a frequency (f) of 31 x 10. Pre-treatment of RBC with dithiothreitol greatly attenuated Kell expression, as expected (data not shown). Among 15 normal adult donors, in each, there was a distinct population of McLeod-like RBC; f ranged from 13.6 x 10 to 113 x 10, with a median of 48.3 x 10 (Figure 1DF). We then tested 17 patients; 13 had “classic” PNH (Table 1). The pattern of Kell and glycophorin-A expression