Differential gene expression in hematopoietic progenitors ... - Nature

Leukemia (2005) 19, 862–868 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu

Differential gene expression in hematopoietic progenitors from paroxysmal nocturnal hemoglobinuria patients reveals an apoptosis/immune response in ‘normal’ phenotype cells G Chen1, W Zeng1, JP Maciejewski1, K Kcyvanfar1, EM Billings2 and NS Young1 1 2

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA; and Bioinformatics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem cell disorder characterized clinically by intravascular hemolysis, venous thrombosis, and bone marrow failure. Despite elucidation of the biochemical and molecular defects in PNH, the pathophysiology of clonal expansion of glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient cells remains unexplained. In pursuit of evidence of differences between GPI-AP-normal and -deficient CD34 cells, we determined gene expression profiles of isolated marrow CD34 cells of each phenotype from PNH patients and healthy donors, using DNA microarrays. Pooled and individual patient samples revealed consistent gene expression patterns relative to normal controls. GPI-AP-normal cells from PNH patients showed upregulation of genes involved in apoptosis and the immune response. Conversely, genes associated with antiapoptotic function and hematopoietic cell proliferation and differentiation were downregulated in these cells. In contrast, the PNH clone of GPI-AP-deficient cells appeared more similar to CD34 cells of healthy individuals. Gene chip data were confirmed by other methods. Similar gene expression patterns were present in PNH that was predominantly hemolytic as in PNH associated with aplastic anemia. Our results implicate an environmental influence on hematopoietic cell proliferation, in which the PNH clone evades immune attack and destruction, while normal cells suffer a stress response followed by programmed cell death. Leukemia (2005) 19, 862–868. doi:10.1038/sj.leu.2403678 Published online 10 March 2005 Keywords: paroxysmal nocturnal hemoglobinuria (PNH); glycosylphosphatidylinositol-anchored protein (GPI-AP); gene expression; microarray

chimeric animals and hematopoietic colony formation from normal and defective embryoid bodies were similar.4–6 While some evidence for intrinsic resistance to apoptosis associated with PIG-A gene mutations has been published,7 these results have not been reproduced in other laboratories.8 Resistance to apoptosis was apparent when BM cells from patients with PNH were separated into GPI-AP-normal and deficient, the ‘normal’ phenotype cells showing increased apoptosis in comparison to normal cells obtained from healthy donors; CD34 cells of the PNH phenotype shared low rates of apoptosis, similar to those measured in healthy volunteer controls.9,10 In order to clarify the selective advantage of the mutant clone in patients with PNH associated with aplastic anemia (AA) as well as in predominantly hemolytic PNH, we have applied DNA chip technology to determine the transcriptomes of patients’ CD34 cells of GPI-AP-normal and -deficient phenotype. Although DNA microarray technology has proven a powerful tool to develop a global and unbiased gene expression profile, small cell numbers may limit the isolation of sufficient RNA required for reproducible analysis.11 Newer small sample protocols that employ RNA amplification of pooled or individual RNA samples have yielded high fidelity and reproducible linear amplification.12–15 Due to the paucity of cells of interest in human BM failure syndromes, we have successfully adapted such protocols to describe the transcriptome of very limited numbers CD34 cells in AA16 and CD34 cells from myelodysplasia characterized by specific chromosomal aberrations.17

Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematologic disease characterized by intravascular hemolysis, venous thrombosis, and bone marrow (BM) failure.1,2 In PNH, somatic PIG-A mutations in a hematopoietic stem cell prevent synthesis of glycosylphosphoinositol (GPI) anchors, which are required for presentation to the cell surface of a large family of proteins. However, PIG-A mutations are necessary but not sufficient to cause PNH disease. Despite brilliant elucidation of the biochemical and molecular lesions in PNH, the explanation for proliferation of the abnormal clone has not been established. PIG-A mutated cells appear to exist in normal individuals but do not expand to represent a significant numbers of peripheral blood granulocytes or erythrocytes.3 In knockout mouse models, PIG-A mutated cells did not give rise to more than a few percentage of circulating progeny in Correspondence: Dr NS Young, National Heart, Lung, and Blood Institute, National Institutes of Health, 9000 Rockville Pike, Bldg 10, 7C/103, Bethesda, MD 20892-1652, USA; Fax: þ 1 301 496 8396; E-mail: [email protected] Received 3 September 2004; accepted 10 January 2005; Published online 10 March 2005

Materials and methods

Patient samples In total, 17 patients with typical clinical features of PNH and evidence of abnormally elevated numbers of GPI-APdeficient circulating granulocytes and erythrocytes were studied (Supplementary data 1); in general, patients suffering predominantly hemolytic PNH reported hemoglobinuria, had high reticulocyte counts and serum lactate dehydrogenase (LDH) levels, but their platelet counts were 480 000/ml; patients with PNH/AA lacked such overt evidence of hemolysis (normal LDH, low reticulocyte counts, and normal urine appearance) and their platelet counts were low. Eight healthy volunteers who were approximately age-matched served as controls. BM was obtained by aspiration from the posterior iliac crest into syringes containing media supplemented 1:10 with heparin (O’Neill and Feldman, St Louis, MO, USA). Informed consent was obtained from all tissue donors according to protocols approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute.

Gene expression profile of CD34 cells in PNH G Chen et al

Phenotypic characterization and separation of GPI-AP-normal and -deficient progenitor cells

Flow cytometry

BM mononuclear cells (BMMNCs) were isolated by density gradient centrifugation using lymphocyte separation medium (Organon, Durham, NC, USA). GPI-AP-normal (CD55 þ CD59 þ ) and GPI-AP-deficient (CD55CD59) CD34 cells were measured and sorted using flow cytometry (Elite ESP, Beckman Coulter, Fullerton, CA, USA). Gates were set based on appropriate isotype controls. The purity of sorted cells ranged from 95–98%.

Fresh BMMNCs from five patients with PNH and three normal controls were stained with FITC-conjugated CD55 and CD59, PerCP-conjugated CD34, and PE-conjugated CD106 (VCAM-1, BD-Pharmingen), or PE-conjugated CD135 (Flt-3, BD-Pharmingen). Using flow cytometry (EPICS Altra, Beckman Coulter, Miami, FL, USA), expression of CD106 and CD135 in the CD55 þ CD59 þ and CD55CD59 cell populations was determined within gates set based on side and forward scatter properties and expression on CD34 cells.

Oligonucleotide microarrays

Results

Due to the very small numbers of cells typically obtained in marrow failure syndromes, in most experiments, RNA was pooled from multiple patients; in some cases, for confirmatory purposes, sufficient RNA could be isolated from individual patients’ BM. The Small Sample Target Labeling protocol of Affymetrix was employed (www.affymetrix.com), version 1 for pooled samples and version 2 for individual patient samples. In total, 500 ng pooled total RNA (an equal quantity of 125 ng of RNA from each patient sample) was used as starting material. For microarray analysis from individual patients, after sorting based on expression of GPI-AP, total RNA was extracted from the normal and deficient CD34 cells with the Small Amount RNA Extracting Kit (Arcturus, Mountain View, CA, USA), followed by two cycles of cDNA synthesis of 200 ng RNA starting material. Image data were analyzed by both Microarray Suite version 5.0 (Affymetrix) and GeneSpring version 6.0 software (Silicon Genetics, Redwood City, CA, USA).

GPI-AP-deficient and -normal CD34 cells in hemolytic PNH and AA/PNH patients

Semiquantitative reverse transcription (RT)-PCR

Characterization of the gene expression profile in PNH patients

Total RNA was prepared from CD34 cells (CD55 þ CD59 þ and CD55CD59) using Trizol reagent as described above. In total 500 ng total RNA was reverse transcribed using the Superscript System (Life Technologies). Samples from six patients (Supplementary data 1), which had not been used in the GeneChip experiments, were assessed by semiquantitative RT–PCR, by use of sequence-specific oligonucleotide primers for the human APO-1, TRAIL, TRAIL receptor DR4, and DR5 genes. The housekeeping gene GAPDH was coamplified as an internal standard control. PCR products were subjected to 1.5% agarose gel electrophoresis.

Quantitative real-time RT–PCR Relative expression levels between GPI-AP-normal and -deficient CD34 cells from individual PNH patients compared to normal controls were validated using the TaqMan 50 nuclease real-time PCR assay. Samples from seven patients (Supplementary data 1) were subjected to real-time RT-PCR using appropriate primers for 12 genes of interest (Supplementary data 2) and b-actin as an internal control. The fold changes in the target genes normalized to b-actin and relative expression of normal control were calculated for each sample using the 2DDCT method, where DDCT ¼ (CT,TargetCT,Actin)Patient’sample (CT,TargetCT,Actin) Normal control.18

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Although patients were classified clinically as predominantly hemolytic or marrow failure variants of PNH, in preliminary experiments we found only minimal differences in the proportion of GPI-AP-normal and -deficient CD34 cells in their BM specimens, as determined by reactivity with antibodies to CD55 and CD59 in flow cytometry. Among hemolytic PNH patients (n ¼ 10), CD34 cells were 58%712 GPI-AP-normal and 42%713 GPI-AP-deficient; in PNH/AA patients (n ¼ 7 for these experiments), CD34 cells were 63%76 GPI-AP-normal and 37%78 GPI-AP-deficient, proportions that were not statistically different between these two diagnostic categories. For further confirmation by micoarray of individual patient samples, we chose stereotypical hemolytic PNH and PNH/AA patients (Supplementary data 1).

For pooled patient samples, GPI-AP-normal and -deficient CD34 cells were obtained from BM aspirations of eight PNH patients (two pools of four patients RNA) and six healthy individuals (two pools of three controls). An additional three individual patient and two control samples also were subjected to microarray analysis. After adjusting for background and noise signals, as defined by an average difference with the arbitrary value of 20, of the 12 000 genes potentially available for analysis, expression of 5400 genes was called as ‘present’. We normalized expression for each chip to the 50th percentile and each gene to median in order to obtain reference values. Differences in expression level using the filters on fold change were then calculated for all individual genes in the test samples from the PNH patients, based on comparison to normal controls. An arbitrary threshold of a three-fold difference was adopted to indicate which genes were differentially expressed. The related expression of these genes was analyzed using a hierarchicalclustering algorithm to identify the pattern of gene expression as a gene tree or condition tree. Nomenclature and functional descriptions were derived from public databases (http://cmap. nci.nih.gov/). Comparison analysis showed similar gene expression profiles of pooled and individual patient samples (Figure 1a), with a large majority of genes over- and underexpressed in both analyses (Figure 1b). Commonly dysregulated genes were selected for annotation. Leukemia


864 were up- or downregulated transcripts in the phenotypically ‘normal’, GPI-AP-positive CD34 cell population (Supplementary data 3). Only a small number of genes in both the GPI-APnormal and -deficient CD34 population from PNH BM appeared to be downregulated in comparison to normal CD34 cells from healthy donors (Supplementary data 4).

Genes upregulated in phenotypically ‘normal’ CD34 cells from PNH patients Gene dysregulation is discussed below by assigned function to gene groups.

Apoptosis genes: The most striking difference between GPIAP-normal and -deficient CD34 cells was in the large number of upregulated apoptosis genes in the phenotypically ‘normal’ population of PNH patients’ cells (Supplementary data 3): among the genes overexpressed in the phenotypically ‘normal’ cells from PNH patients were APO-1, TRAIL, TRAIL receptor DR4, and DR5; all have activity in inducing programmed cell death through activation of the Fas-associated pathway, leading to apoptosis.19,20 Cytokines and chemokines: Many upregulated genes in

Figure 1 Gene expression profile of GPI-AP-normal and -deficient CD34 cells in PNH relative to CD34 cells from normal controls. The expression pattern of genes is displayed in hierarchical-clustering format, where rows indicate experimental samples and columns represent genes. Pooled: pooled samples; individuals: individual samples. (a) Global gene expression pattern. Each value represents the difference from the gene median and is depicted according to the color scale from green (underexpression) to red (overexpression). Black indicates average expression. (b) The distribution of dysregulated gene expression in GPI-AP-normal and -deficient CD34 cells of pooled (n ¼ 8 patients) and individual samples (n ¼ 3 patients) with PNH compared to normal. In GPI-AP-normal CD34 cells, 96 and 84 genes were identified as commonly up- and downregulated expression in pooled and individual samples; in GPI-AP-deficient CD34 cells, 59 and 56 genes were over- and underexpression in both pooled and individual samples.

Gene expression pattern of GPI-AP-normal and -deficient cells from PNH patients and normal cells from healthy donors Gene activity in populations of CD34 cells from PNH patients separated based on the presence of GPI-AP on the cell surface could be related to the normal gene expression pattern of CD34 cells from healthy donors; surprisingly, the pattern of overall gene expression that more closely resembled true normal cells was present in the PNH clone of GPI-AP-deficient CD34 cells from the patients (Figure 1). Only a very limited number of genes was observed to be dysregulated in CD34 cells derived from the PNH clone, in comparison to CD34 cells from healthy donors (including, as examples, CCTZ-2, CA2, HDC, GLUT1, CLG, SNRPEP1, TRF1, and RIP140). As described below, the great majority of abnormally expressed genes in the patient samples Leukemia

the GPI-AP-normal CD34 cells from PNH BMs were members of families of inflammatory cytokine and immunoregulatory response genes, including interleukins, and class II major histocompatibility complex (MHC) molecules (Supplementary data 3). Tumor necrosis-factor-receptor-1A (TNFR1A) and multiple interferon-inducible genes, including interferon-gamma (IFN-g) and interferon-inducible T-cell alpha chemokine (I-TAC), were also increased in expression in the GPI-AP-normal cells from PNH patients. The cytokines and chemokines encoded by these genes play roles in the regulation of immune and inflammatory responses as well as in hematopoietic suppression.21–23 Several adhesion molecules showed increased expression in the phenotypically ‘normal’ CD34 cells from PNH patients’ BMs, such as vascular adhesion molecule-1 (VCAM-1) gene. VCAM-1 has been implicated in local inflammation in the pathophysiology of some autoimmune diseases, and its upregulation is consistent with increased expression of TNF and TNFR1.

Cell cycle control genes: Also in the GPI-AP-normal CD34 cells in PNH, we detected overexpression of a number of cell cycle inhibitory genes (WAF, p21, and KIP2). These genes belong to a family of cell cycle-dependent kinase inhibitors, which modulate cell growth, differentiation, and apoptosis.24,25 Cell surface signal transduction genes: Among the genes in this class overexpressed in the ‘normal’ CD34 cells of PNH patients’ BMs were signaling lymphocyte activation molecule (SLAM or CD150), Wilms tumor-1 (WT1) gene, signal transducers and activators of transcription-1 and -4 (STAT1, STAT4), and early growth response genes EGR2 and EGR3, which play a role in the regulation of hematopoietic cell proliferation and differentiation.26–30

Genes related to complement activation and blood coagulation: Owing to the proclivity of patients with PNH to thrombosis in unusual venous sites, we were interested in expression of genes related to the blood coagulation pathway as


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Figure 2 Gene expression analyses by semiquantitative RT-PCR. GPI-AP-normal and -deficient CD34 cells were isolated from BMMNCs of six PNH patients. All samples were amplified with the appropriate primers as described. Amplification products were separated on a 1.5% agarose gel and stained with ethidium bromide. The relative intensity of each band was quantitated by imaging. nl: normal control; þ : GPI-AP-normal CD34 cells from PNH patient; : GPI-AP-deficient CD34 cells.

well as to complement activation. C1QB is a complement component in the classical pathway of activation recognized by immunoglobubin, and this gene showed increased expression in the phenotypically ‘normal’ CD34 cells of the PNH patients. A number of genes implicated in hemostasis and thrombosis, including APOE, ADORA2A and coagulation factor III (F3), heparin binding factor III, were upregulated in the GPI-APnormal cell population.

Genes downregulated in phenotypically ‘normal’ CD34 cells from PNH patients A limited number of genes showed downregulated expression specifically in GPI-AP-normal CD34 cells from PNH patients (Supplementary data 3); in general, their function opposed genes upregulated in this cell population. As genes that promote apoptosis, proliferation and differentiation were upregulated in these cells, antiapoptosis genes were downregulated, including DFF45, Flt-3, STK-1, E2F, and DP-I. STK-1 binds FLT-3 to initiate the FLT3 signaling pathway, important in proliferation of hematopoietic stem cells and development of B cell progenitors, NK cells, and dendritic cells; the FLT3 pathway also has roles in blocking differentiation and apoptosis and in leukemogenesis.31,32

Genes dysregulated in both GPI-AP-normal and -deficient cells in PNH patients, compared to healthy control CD34 cells Only a few genes were overexpressed in both GPI-AP-normal and -deficient CD34 cells of PNH patients’ BMs, in comparison to healthy donors’ CD34 cells. Of genes downregulated in these two CD34 cells of PNH patients’ BMs (Supplementary data 4) were some related to the growth and function of platelets, and genes that encoded proteins involved in wound repair, inflammation, and coagulation, such as platelet factor 4 (PF4), CD41b, CD62 (selectin P), and proplatelet basic protein (PPBP). Important components of crucial transduction pathways were also downregulated in PNH, including both the c-myb and

c-myc genes. Expression of c-myb also is decreased in normal and leukemic cell terminal differentiation, and its blockade inhibits proliferation of normal hematopoietic precursors in vitro.33

Validation of microarrary gene expression results To confirm the DNA GeneChip data, the expression of mRNA was analyzed for APO-1, TRAIL, and TRAIL receptors (DR4 and DR5) by semiquantitative RT-PCR analysis (Figure 2). For this purpose, GPI-AP-normal and -deficient CD34 cells were obtained from a further six individual PNH patients who had not contributed BM for the GeneChip experiments. Expression of all the implicated genes in the apoptosis pathways were lower in the GPI-AP-deficient CD34 cells from these patients than in the phenotypically ‘normal’ cells, and similar in expression levels to normal CD34 cells obtained from healthy donors, confirming the microarray experiments. Further validation was obtained by real-time RT-PCR using RNA from GPI-AP-normal and -deficient CD34 cells from seven individual PNH patients (four hemolytic PNH and three PNH/ AA patients; only one patient had contributed to the DNA GeneChip pooled samples). Relative expression levels initially determined with the microarrary were correlated with TaqMan results for a subset of individual samples. We chose APO-1, TRAIL, TRAIL-DR4, TRAIL-DR5, IL-1B, IL-6, IL-7, IL-8, TNF-a, and IFN-g, which were upregulated, and STK-1, which was downregulated, in GPI-AP-normal CD34 cells from PNH patients in comparison with GPI-AP-deficient CD34 cells from PNH patients and to CD34 cells from healthy donors. In addition, the PF4 gene, which appeared downregulated in both GPI-AP-normal and -deficient CD34 cells from PNH patients compared to normal controls, was also assayed. The high correlation between the two methods for all 12 genes in all the individual PNH patients confirmed the identification of dysregulated genes by microarray (Figure 3a and b). We further analyzed the expression level of specific genes in individual hemolytic PNH and PNH/AA patients: the similar gene expression pattern in GPI-AP-normal or -deficient CD34 cells in both clinical variants (Figure 3c) suggested no significant Leukemia


866 difference in transcription associated with the clinical features of red cell destruction or cytopenias. As further validation of the microarray method, cell surface expression of VCAM-1 (CD106) and Flt3 (CD135) was examined by flow cytometry. CD106 was expressed at low level in GPI-AP-deficient CD34 cells (0.470.2) from five PNH patients and in control CD34 cells (0.570.3) from three healthy

volunteers, while a higher proportion of CD34 cells with ‘normal’ phenotype (5.972.1) derived from PNH patients expressed CD106 (Supplementary data 5a and c). CD135 was detected in a lower number of GPI-AP-normal CD34 cells (7.673.2) from PNH patients, and a much higher proportion of CD34 cells (3576.5) from healthy volunteers and in GPI-APdeficient CD34 cells (3778.9) from PNH patients (Supplementary data 5b and d).

Discussion DNA microarray technology has allowed us to assess the expression levels of thousands of genes in GPI-AP-normal and -deficient CD34 cells from PNH patients, in comparison of the two populations to each other as well as to CD34 cells from healthy donors. Due to the very limited numbers of GPI-APnormal and -deficient CD34 cells in PNH patients’ BMs, and in order to obtain adequate amounts of RNA for reproducible microarrary analyses, we used pooled samples and the T7-based linear RNA amplification method. Replicates for pooled samples showed excellent correlations in scatter plots. Furthermore, there was generally good agreement between pooled sample data and results of microarray analysis performed for individual patients’ RNA. To assure that the use of pooled samples and amplified RNA accurately preserved differences among samples, we chose 14 genes of interest for further measurement using semiquantitative PCR, real-time PCR, and flow cytometry to verify expression levels in samples of unamplified RNA or whole cells from individual patients. The excellent correspondence of results on linear regression analysis indicated some reliability for our strategy of gene expression profiling. Overall, the gene expression pattern in PNH patients’ BMs was surprising in that the phenotypically ‘normal’ cells that express GPI-AP were more markedly abnormal than was the pattern in the GPI-AP-deficient cells derived from the mutant PNH clone. PNH stem cells appeared to have a growth advantage when inoculated into immunodeficient host mice,34 and we and others have noted preferential growth of GPI-AP deficient cells in tissue culture; in these experiments, cells of normal phenotype but not the mutant clone aberrantly expressed Fas and annexin.9,10 As discussed below, the increased expression of genes related to apoptosis and the immune response supports the currently preferred model of PNH clonal expansion as a result of environmental selection of pre-existing quiescent mutant cells.35 When individual genes were assayed in CD34 cell samples of individual patients or on comparison of microarray data obtained from individual patients, we also did not observe any major Figure 3 Relative gene expression levels by real-time RT-PCR. The fold-difference in expression of target genes relative to an internal control gene (b-actin) was studied using the 2DDCT method. In total 12 genes of interest were assessed in GPI-AP-normal and -deficient CD34 cells from seven PNH patients, including four hemolytic PNH and three PNH/AA. (a) The average fold-change of 12 genes’ expression. (b) Change of gene expression level was calculated as fold-change compared to normal control by both real-time RT-PCR and microarray assay. Real-time PCR data were highly correlated to the GeneChip data by linear regression analysis. (c) Fold-changes in expression of 12 genes from individual hemolytic PNH and PNH/AA patients. Similar gene expression patterns of GPI-AP-normal or -deficient CD34 cells were found in both diagnostic categories. J: GPI-AP-deficient CD34 cells from hemolytic PNH patients; K: GPI-AP-deficient CD34 cells from PNH/AA patients; n: GPI-AP-normal CD34 cells from hemolytic PNH; m: GPI-AP-normal CD34 cells from PNH/AA patients PNH. Leukemia


867 difference in specific or global gene expression for CD34 cells of hemolytic PNH and PNH/AA. Earlier work has shown that hematopoietic colony formation is poor in PNH, irrespective of either marrow cellularity or the presence of gross hemolysis. Clinically, patients may show evolution from marrow failure to hemolytic anemia years following immunosuppressive therapy, and patients presenting with hemolytic symptoms often progress to AA.36 Most striking in the current experiment was high expression of apoptosis genes in phenotypically ‘normal’ CD34 cells of PNH patients. Apoptosis can be triggered through a variety of systems, and our data would implicate several major mechanisms, including TNF and its receptors, Fas ligand and Fas, and the perforin-granzyme system. We and others have previously reported high expression of cytokines that trigger Fas-mediated apoptosis, especially overexpression of IFN-g and TNF. Both these genes also were overexpressed in the GPI-AP-normal cells of the PNH patients. Although the purity of our CD34 cells was on average high, contamination of less wellfractionated donor samples might have allowed inclusion of small numbers of T cells. Whether the detection of these genes is indicative of production of these cytokines by CD34 cells (a capability suggested in some experiments37) or small number of T cells physically associated with CD34 cell targets remains uncertain and the subject of further investigation. Nevertheless, the GPI-AP-normal CD34 cells in PNH patients showed abundant evidence of a variety of signal transduction pathways activated by engagement of IFN-g and TNF, regardless of whether these cytokines were of paracrine or autocrine origin. Microarray technology is a powerful method of hypothesis generation. In PNH, the often fatal proclivity towards venous thrombosis in unusual anatomic sites remains without satisfactory explanation. We observed a number of genes related to platelet development and function to be aberrantly expressed, which may offer clues as to the origins of the prominent thrombophilia. Indeed, we may possibly have underestimated genes of interest but only modestly dysregulated, due to the necessary adoption of arbitrary thresholds for purposes of comparison and statistical analysis. In more limited experiments using arrays to analyze granulocytes in PNH, decreased expression of EGR1 and TAXREB107 was noted in normal white blood cells;38 we observed similar differences but not to levels that were considered statistically significant. The pattern of gene expression in GPI-AP-normal cells in PNH suggests not only a ‘stress’ response to immune system effector cells and molecules but also general inhibition of cell cycling and proliferation, due either to abnormalities in the intrinsic cell program or as a result of immune system attack and induction of apoptosis. However, data obtained in primary hematopoietic cells greatly differ from our earlier findings in preliminary experiments using paired leukemic cells lines and paired virustransformed lymphoblastoid cell lines derived from patients (Miyazato et al. Blood 200l;78:222a, abstract). In these cell lines, remarkably few genes differed in expression between cells separated by GPI-AP expression, a strong argument against an intrinsic alteration in cellular programs as a simple consequence of the PIG-A gene mutation (as for example resistance to apoptosis as has been suggested by one group8). In contrast, the abundant data derived from the current microarray analysis, combined with murine experiments, the observation of small numbers of PNH cells in normal humans, and the functional behavior of patients’ marrow cells in vitro, all support a model of PNH clonal escape from immune attack and destruction in T-cell mediated BM failure.

Supplementary Information Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu).

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