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Very few human genes can be used to identify spontaneous inactivating somatic mutations. We hypothesized that because the XK gene is X-linked, it would be easy to identify spontaneously arising red cells with a phenotype resembling the McLeod syndrome, which results from inherited XK mutations. Here, by flow cytometry, we detect such phenotypic variants at a median frequency of 9 × 10−6 in neonatal cord blood samples and 39 × 10−6 in healthy adults (p = 0.004). It may be possible to further investigate the relationship between aging, mutations, and cancer using this approach.
Spontaneous inactivating somatic mutations are a key process in malignant transformation. The vast majority of mutations, however, will not be detectable because there is either no phenotype or, conversely, the mutations are lethal. Furthermore, for most human genes there are two active copies, and the chance of identifying a cell that has lost the function of both alleles would probably be so low as to preclude detection. Due to these considerations, there are only a handful of genes in which somatic mutations can be identified in normal human cells.
The most commonly used gene, HPRT, is on the X-chromosome; therefore, only a single mutation is required to produce the mutant phenotype, due to hemizygosity in males and X-inactivation in females. HPRT mutant lymphocytes, which have impairment in the purine salvage pathway, are detected by their ability to grow in the presence of 6-thioguanine in limiting dilution cloning [1,2]. A different approach is based on GPA or HLA-A. These genes are autosomal, but they are polymorphic, and in individuals with certain genotypes, phenotypic variants with spontaneous loss of a single allele can be detected [3-10]. The assay for spontaneously arising GPA variants is particularly robust due to the ease of obtaining red cells. However, it is suitable only for compound heterozygotes carrying both the M and N alleles.
Our previous work [11-13] has been based on the PIG-A gene , which is X-linked, like HPRT. As for HLA and GPA, PIG-A mutations affect the surface of the cell, in this case, resulting in a lack of glycosylphosphatidylinositol (GPI) linked proteins . As a result, mutants can be detected by flow cytometry, which provides a very rapid screen for rare GPI (−) leucocytes. Red cells can be analyzed as well , but unlike for leucocytes, complement-mediated hemolysis will select against GPI (−) red cells in vivo .
Hoping to find other examples of genes with the same favorable characteristics as PIG-A, we performed a literature search for other X-linked genes that affect the red cell membrane, and XK (Xp21.1) emerged as a prime candidate. Like HPRT, much is known about the XK gene because inherited mutations are associated with a known human genetic condition, the McLeod syndrome [16-18]. Affected males with this condition are noted to have some acanthocytes on the peripheral blood smear , and they have a mild compensated hemolysis with generally normal hemoglobin levels [18,19]. There is an absence of XK antigens on the red cell surface, and there is a great reduction in Kell protein levels . This occurs because the Kell protein is covalently linked to the XK protein by a disulfide bond, which is required for the expression of Kell proteins on the surface . [Although there are polymorphic determinants on the Kell protein that are important in blood banking, including the “Kell” (Met193) and “Cellano” (Thr193) blood group antigens, here “KEL” refers to the gene on chromosome 7, and “Kell protein” refers to its 732 amino acid gene product, regardless of these polymorphisms.] Obligate female heterozygote carriers have two populations of red cells with respect to this phenotype, due to random X-chromosome inactivation [18,20]. Some affected males will also develop muscular atrophy, neuropathy, and psychiatric changes [20,21], presumably due to loss of an unknown function of XK on the surface of myocytes and neurons.
The XK gene was cloned in 1994 , it is about 12 MB centromeric to the PIG-A gene, 4 MB centromeric to DMD, and immediately upstream of CYBB, and its gene product is a 444 amino acid surface protein, which spans the membrane 10 times [17,22-24]. Among over 150 reported cases of the McLeod syndrome , at least 28 mutations have been identified [26,27]. These include frameshift, nonsense, missense and splice site mutations, partial deletions, and large deletions of over 5.5 MB [20,26-28]. Neighboring genes on the X-chromosome can be simultaneously affected by large deletions involving XK, resulting in muscular dystrophy  or chronic granulomatous disease [29,30].
We have postulated that just as XK mutations can be present in the germline of affected individuals, somatic XK mutations might be present in erythropoietic cells in normal individuals. Unlike the case for PIG-A mutant red cells, hemolysis is mild in the McLeod syndrome [18,19], so we predicted that spontaneously arising red cells with the XK null phenotype would not be significantly selected against in vivo. Using an antibody that is specific for a non-polymorphic Kell protein determinant, we have now detected McLeod-like red cells in normal individuals. Here we compare the frequency of these phenotypic variants in adults and umbilical cord blood samples as well as with results from the other assay systems.
Umbilical cord blood samples from placentas were collected, and whole blood samples from healthy consenting adults were obtained, in accordance with IRB protocols. K14 antibody recognizes a non-polymorphic antigen (Arginine 180) on the Kell protein that is expressed on red cells from all donors . Thawed aliquots of red cells from a patient with the McLeod syndrome and an obligate heterozygote carrier female served as controls. Approximately 10 to 15 million red cells were first incubated on ice with undiluted K14 hybridoma supernatant (New York Blood Center) for 30 minutes at 108 cells/ml and washed twice. The cells were then incubated with a 1:5 dilution of PE-conjugated rabbit-anti-mouse antibody (Dako-Cytomation), washed twice again, followed by incubation with a FITC-conjugated antibody specific for a non-polymorphic glycophorin A determinant (Dako-Cytomation), at a 1:10 dilution. Cells were washed again and passed through a 35 μM filter prior to analysis on a Becton-Dickinson FACScan. Forward and side scatter were acquired on a log-log scale, and only glycophorin A (+) events were gated. At least 1.2 million gated events were collected.
Normal red cells expressed glycophorin A as well as the Kell protein (figure 1A, left panel). Red cells from a patient with the McLeod syndrome expressed significantly lower levels of Kell proteins but normal levels of glycophorin A (figure 1A, middle panel). As expected, given random X-chromosome inactivation, a female obligate carrier of the McLeod syndrome had two populations, in about a 1:1 ratio, suggesting lack of significant selection against the mutants in vivo (figure 1A, right panel).
In 8 adult normal donors, there was a small population of red cells with a McLeod -like phenotype (figure 1B), at a median frequency of 39 × 10−6 (range 26 to 61 × 10−6). As a comparison, we also measured the frequency of cells with a PIG-A mutant phenotype by staining red cells with a mixture of anti-CD59 and anti-CD55 antibodies, followed by rabbit anti-mouse-PE and then anti-glycophorin A antibodies as previously described . In 5 adults, the frequency of red cells with the PIG-A mutant phenotype (range 0.8 to 6.3 × 10−6, mean 3.3 × 10−6) was considerably lower than the frequency of McLeod-like cells, which is likely accounted for due to lysis of GPI (−) red cells. We hypothesized that somatic XK mutations in erythrocyte progenitors and stem cells are lowest at birth and accumulate with age. We therefore performed a parallel analysis of red cells derived from umbilical cord blood samples. Indeed, in 5 cord blood samples there was ~4 fold lower frequency of McLeod-like red cells-- a median of 9 × 10−6, (range 6.9 to 16.3 × 10−6; p = 0.004, two-sided Mann Whitney test, figure 1D, table 1).
In patients with the McLeod syndrome, about 25% of the red cells have membrane projections , and we tried two approaches to isolate these cells for visualization: sorting to collect the K14 negatives, and MiniMacs bead depletion of the K14 positive cells. In some cases, we were able to identify membrane projections in the enriched population by light microscopy (figure 1E). We also incubated red cells with K14 antibody, performed bead depletion, followed by sequential staining with rabbit anti-mouse PE and anti-glycophorin A-FITC, followed by Compucyte iCyte analysis. The gated FITC-positive, PE-negative (McLeod-like) population appeared as deeply green staining discocytes, whereas incidentally visualized neighboring cells and gated FITC (+) PE (+) cells produced a merged yellow fluorescence (figure 1F). While membrane projections were not seen in this experiment, these abnormalities might not be preserved during the incubation and selection steps, perhaps as a result of changes in phospholipids, which can affect the acanthocyte phenotype .
Here we have shown that it is possible to identify in normal individuals phenotypic variant red cells resembling cells from patients with the McLeod syndrome. As for assays of the frequency of GPA variants, the analysis of McLeod-like cells has as a disadvantage the fact that DNA can not be extracted from normal red cells for sequencing. We believe that it may be possible to address this question in the future using samples from individuals who, as a result of pathological conditions, have large numbers of circulating nucleated red cells. As for analyses based on GPA, an advantage of this assay is the ease of isolation of red cells, the large number of cells that can be screened by flow cytometry, and their uniform size distribution, which facilitates flow cytometry. An additional advantage of the analysis for McLeod-like cells is that it can be performed on samples derived from any individual, whereas only compound heterozygotes carrying both the M and N alleles can be studied in the GPA analysis. We do not yet know whether cells from laboratory animals can be studied in this way. However, it is notable that Xk is on the mouse X-chromosome. As in humans, the mouse Xk protein is covalently linked to the gene product of the mouse KEL homologue, and both are expressed on red cells [33-35].
The median frequency of phenotypic variants for normal adults in this study, 39 × 10−6, is higher than corresponding values for 6-thioguanine resistant lymphocytes (1.9 to 8.7 × 10−6 [1-3]), GPI (−) granulocytes (22 × 10−6, ), GPI (−) marrow precursors (14.7 × 10−6 ), GPI (−) lymphocytes (18 × 10−6, ), GPA MN variant red cells (7 to 10 × 10−6 [8,10]), and HLA-A variant lymphocytes (24 × 10−6 ) in normal adults. A possible explanation for this difference would be if red cell progenitors could tolerate larger deletions involving XK compared with these other genes. It is also possible that mutations in both copies of the KEL gene (which is autosomal) could produce the same phenotype, but we believe that this is less likely to occur than a single mutation in XK. We can not rule out that in some cells, global abnormalities in the synthesis of membrane proteins would mimic the McLeod phenotype. However, we believe that gating to include only glycophorin-A positive cells would likely exclude such events.
Others have observed that the frequency of phenotypic variants is higher in adults than in neonates [3,5,38-40]. In one study, the median frequency of lymphocytes with the HPRT mutant phenotype was about 1 × 10−6 in neonates and it increased with a slope of about 0.2 × 10−6 per year through adulthood . In a separate study, the log of the frequency of phenotypic variants appeared to be increasing linearly, with a multiplicative increase of 1.6% per year . In a study of spontaneous HLA-A allele loss, the frequency of phenotypic variants increased from a geometric mean of 7 × 10−6 in cord blood samples to 24 × 10−6 in 20 to 60 year olds, and 65 × 10−6 in those over the age of 60, with a suggestion of exponential increases in the oldest subjects . In contrast, no changes were seen in the frequency of spontaneous GPA allele loss variants between cord blood samples and adolescents .
As in these previous studies using HPRT and HLA-A, our data demonstrates that the frequency of spontaneously arising XK phenotypic variants is higher in adults than neonates. This could be due to accumulation of mutations or an increase in the rate of mutations with age, as we do not yet know whether the increase is linear or exponential. A second question is whether differences in fetal and adult hematopoiesis, including migration of stem cells and fetal to adult hemoglobin gene switching, could somehow be responsible for the lower frequency of phenotypic variants in neonates. We believe that a detailed study of elderly individuals compared with those of different age ranges will address these two questions. Furthermore, it may be possible to investigate the relationship between aging and cancer risk with this approach.
Supported in part by NIH grants RO1 CA-109258 (DJA) and RO1 HL075716 (SL). We are indebted to Dr. Marion Reid of the New York Blood Center for assistance in obtaining control red cell samples, Gregory Halverson of the New York Blood Center for assistance in obtaining K14 hybridoma supernatant, and to Bridget Lane of the NYU Cancer Center for assistance in coordinating the collection of blood samples from healthy donors. We would like to thank Dr. Ed Luther for assistance with the iCyte analysis.
Conflict of Interest Disclosures: The authors do not have any conflicts of interest to disclose
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