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Hypoxia is emerging as an important characteristic of the hematopoietic stem cell (HSC) niche, but the molecular mechanisms contributing to quiescence, self-renewal, and survival remain elusive. Vascular endothelial growth factor A (VEGFA) is a key regulator of angiogenesis and hematopoiesis. Its expression is commonly regulated by hypoxia-inducible factors (HIF) that are functionally induced in low-oxygen conditions and that activate transcription by binding to hypoxia-response elements (HRE). Vegfa is indispensable for HSC survival, mediated by a cell-intrinsic, autocrine mechanism. We hypothesized that a hypoxic HSC microenvironment is required for maintenance or up-regulation of Vegfa expression in HSCs and therefore crucial for HSC survival. We have tested this hypothesis in the mouse model Vegfa(δ/δ), where the HRE in the Vegfa promoter is mutated, preventing HIF binding. Vegfa expression was reduced in highly purified HSCs from Vegfa(δ/δ) mice, showing that HSCs reside in hypoxic areas. Loss of hypoxia-regulated Vegfa expression increases the numbers of phenotypically defined hematopoietic stem and progenitor cells. However, HSC function was clearly impaired when assessed in competitive transplantation assays. Our data provide further evidence that HSCs reside in a hypoxic microenvironment and demonstrate a novel way in which the hypoxic niche affects HSC fate, via the hypoxia-VEGFA axis.

Foamy virus (FV) vectors are particularly attractive gene-transfer vectors for stem-cell gene therapy because they form a stable transduction intermediate in quiescent cells and can efficiently transduce hematopoietic stem cells. Here, we studied the use of FV vectors to transduce long-term hematopoietic repopulating cells in the dog, a clinically relevant large animal model. Mobilized canine peripheral blood (PB) CD34+ cells were transduced with an enhanced green fluorescent protein (EGFP)-expressing FV vector in an 18-hour transduction protocol. All 3 dogs studied had rapid neutrophil engraftment to greater than 500/microL with a median of 10 days. Transgene expression was detected in all cell lineages (B cells, T cells, granulocytes, red blood cells, and platelets), indicating multilineage engraftment of transduced cells. Up to 19% of blood cells were EGFP+, and this was confirmed at the DNA level by real-time polymerase chain reaction (PCR) and Southern blot analysis. These transduction rates were higher than the best results we obtained previously with lentiviral vectors in a similar transduction protocol. Integration site analysis also demonstrated polyclonal repopulation and the transduction of multipotential hematopoietic repopulating cells. These data suggest that FV vectors should be useful for stem-cell gene therapy, particularly for applications in which short transduction protocols are critical.

Knowledge of the composition and interrelationship of the various hematopoietic stem cells (HSCs) that comprise the human HSC pool and the consequence of culture on each class is required for effective therapies based on stem cells. Clonal tracking of retrovirally transduced HSCs in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice revealed heterogeneity in the repopulation capacity of SCID-repopulating cells (SRCs). However, it is impossible to establish whether HSC heterogeneity is intrinsic or whether the culture conditions required for retroviral transduction induce qualitative and quantitative alterations to SRCs. Here, we report establishment of a clonal tracking method that uses lentivectors to transduce HSCs with minimal manipulation during overnight culture without cytokine stimulation. By serial bone marrow (BM) sampling of mice receiving transplants, short-term SRCs (ST-SRCs) and long-term SRCs (LT-SRCs) were identified on the basis of repopulation dynamics demonstrating that their existence is not an experimental artifact but reflects the state of the HSC pool. However, 4 days of culture in conditions previously used for SRC retroviral transduction significantly reduced SRC number as assessed by clonal analysis. These studies provide a foundation to understand the molecular and cellular determinants of human HSC development and to develop therapies targeted to specific HSC classes.

For the treatment of beta-globin gene defects, a homologous recombination-mediated gene correction approach would provide advantages over random integration-based gene therapy strategies. However, "neighborhood effects" from retained selectable marker genes in the targeted locus are among the key issues that must be taken into consideration for any attempt to use this strategy for gene correction. An Ala-to-Ile mutation was created in the beta6 position of the mouse beta-major globin gene (beta(6I)) as a step toward the development of a murine model system that could serve as a platform for therapeutic gene correction studies. The marked beta-major gene can be tracked at the level of DNA, RNA, and protein, allowing investigation of the impact of a retained phosphoglycerate kinase (PGK)-neo cassette located between the mutant beta-major and beta-minor globin genes on expression of these 2 neighboring genes. Although the PGK-neo cassette was expressed at high levels in adult erythroid cells, the abundance of the beta(6I) mRNA was indistinguishable from that of the wild-type counterpart in bone marrow cells. Similarly, the output from the beta-minor globin gene was also normal. Therefore, in this specific location, the retained, transcriptionally active PGK-neo cassette does not disrupt the regulated expression of the adult beta-globin genes. (Blood. 2001;98:65-73)

Recent experiments point to the great value of lentiviral vectors for the transduction of human hematopoietic stem cells (hHSCs). Vectors used so far, however, have been poorly satisfying in terms of either biosafety or efficiency of transgene expression. Herein is described the results obtained with human immunodeficiency virus-based vectors optimized in both of these aspects. It is thus shown that vectors containing the EF1alpha and, to a lesser extent, the phosphoglycerate kinase (PGK) promoter, govern high-level gene expression in human hematopoietic progenitors as well as derived hematopoietic lineages of therapeutic relevance, such as erythrocytes, granulocytes, monocytes, dendritic cells, and megakaryocytes. EF1alpha promoter-containing lentiviral vectors can also induce strong transgene expression in primary T lymphocytes isolated from peripheral blood. A self-inactivating design did not affect the performance of EF1alpha promoter-based vectors but significantly reduced expression from the PGK promoter. This negative effect could nevertheless be largely rescued by inserting the post-transcriptional regulatory element of woodchuck hepatitis virus upstream of the vector 3' long terminal repeat. These results have important practical implications for the genetic treatment of lymphohematologic disorders as well as for the study of hematopoiesis via the lentivector-mediated modification of hHSCs.

A novel glass needle-mediated microinjection method for delivery of macromolecules, including proteins and larger transgene DNAs, into the nuclei of blood stem/progenitor cells was developed. Temporary immobilization of cells to extracellular matrix-coated dishes has enabled rapid and consistent injection of macromolecules into nuclei of CD34(+), CD34(+)/CD38(-), and CD34(+)/CD38(-)/Thy-1(lo) human cord blood cells. Immobilization and detachment protocols were identified, which had no adverse effect on cell survival, progenitor cell function (colony forming ability), or stem cell function (NOD/SCID reconstituting ability). Delivery of fluorescent dextrans to stem/progenitor cells was achieved with 52% +/- 8.4% of CD34(+) cells and 42% +/- 14% of CD34(+)/CD38(-)cells still fluorescent 48 hours after injection. Single-cell transfer and culture of injected cells has demonstrated long-term survival and proliferation of CD34(+) and CD34(+)/CD38(-) cells, and retention of the ability of CD34(+)/CD38(-) cells to generate progenitor cells. Delivery of DNA constructs (currently

Stem cell factor (SCF) is expressed as an integral membrane growth factor that may be differentially processed to produce predominantly soluble (S) (SCF(248)) or membrane-associated (MA) (SCF(220)) protein. A critical role for membrane presentation of SCF in the hematopoietic microenvironment (HM) has been suggested from the phenotype of the Steel-dickie (Sl(d)) mice, which lack MA SCF, and by studies performed in our laboratory (and by others) using long-term bone marrow cultures and transgenic mice expressing different SCF isoforms. Steel(17H) (Sl(17H)) is an SCF mutant that demonstrates melanocyte defects and sterility in males but not in females. The Sl(17H) allele contains a intronic mutation resulting in the substitution of 36 amino acids (aa's) in the SCF cytoplasmic domain with 28 novel aa's. This mutation, which affects virtually the entire cytoplasmic domain of SCF, could be expected to alter membrane SCF presentation. To investigate this possibility, we examined the biochemical and biologic properties of the Sl(17H)-encoded protein and its impact in vivo and in vitro on hematopoiesis and on c-Kit signaling. We demonstrate that compound heterozygous Sl/Sl(17H) mice manifest multiple hematopoietic abnormalities in vivo, including red blood cell deficiency, bone marrow hypoplasia, and defective thymopoiesis. In vitro, both S and MA Sl(17H) isoforms of SCF exhibit reduced cell surface expression on stromal cells and diminished biological activity in comparison to wild-type (wt) SCF isoforms. These alterations in presentation and biological activity are associated with a significant reduction in the proliferation of an SCF-responsive erythroid progenitor cell line and in the activation of phosphatidylinositol 3-Kinase/Akt and mitogen-activated protein-Kinase signaling pathways. In vivo, transgene expression of the membrane-restricted (MR) (SCF(X9/D3)) SCF in Sl/Sl(17H) mutants results in a significant improvement in peripheral red blood cell counts in comparison to Sl/Sl(17H) mice.

A new assay is described that measures the numbers (n) of endogenous long-term repopulating cells (eLTRC) surviving sublethal irradiation. The assay is based on analysis of variances of Pgk-1 phenotypes within groups of sublethally irradiated Pgk-1(a/b) mice. When ln n is plotted as a function of dose, the radiosensitivity (D0) is the negative reciprocal of the slope and the gamma-intercept is N or total numbers of eLTRC per mouse. The assay is unique in that N is an absolute value not requiring correction for seeding efficiency, or f. From two independent estimates, total numbers of eLTRC were determined to be 8,700 or 11,900 cells per mouse and D0 was found to be 0.82 or 0.83 Gy. If eLTRC are, in fact, the long-term repopulating cells (LTRC) defined by classical transfer assay, then LTRC can home to the marrow with nearly unit efficiency, only one to two LTRC are required for a mouse to survive a radiation LD50/30, and LTRC possess nearly unlimited self-renewal potential.

The utility of X-inactivation based clonality assays for evaluation of human neoplasia is well-documented. However, excessive Lyonization is a potential limitation of these assays, because it mimics clonal derivation of cells. The incidence of excessive Lyonization in healthy females is controversial, with reported incidence varying from 4% to 33%. Several explanations have been offered for the observed variation, including different criteria for excessive Lyonization, diversity of X-linked clonality assays, small population sizes and more recently, tissue specificity of X-inactivation patterns. However, it is also possible that stem cell depletion, clonal hematopoiesis, or selection pressures on blood cells results in an increased incidence of excessive skewing. If any of the latter is true, then the incidence of excessive skewing should increase with age in blood cells. To test this hypothesis, we determined X-inactivation ratios at the human androgen receptor locus of 295 normal females from three age groups: (1) neonates, (2) females 28 to 32 years old and, (3) females aged > or = 60 years. The incidence of skewing (allele ratios > or = 3:1) was 8.6% (14 of 162) in neonates (P = .104 v 28 to 32); 16.4% (11/67) in 28 to 32 y.o. (P = .0064 v > or = 60), and 37.9% (25 of 66) in women > or = 60 y.o. (P < .0001 v neonates). When a more stringent criterion for skewing was used (allele ratios > or = 10:1), the incidence was 1.9% (3 of 162) in neonates (P = .362 v 28 to 32), 4.5% (3 of 67) in 28 to 32 y.o. (P = .0022 v > or = 60), and 22.7% (15 of 66) in > or = 60 y.o. group (P < .0001 v neonates). Results have been confirmed at the phosphoglycerate kinase locus for 48 heterozygous females. The incidence of excessive skewing increases with age. In neonates, the incidence is low and may correspond to true excessive Lyonization. Acquired skewing occurs with aging in normal females and is present in 38% of females over the age of 60. Further studies are needed to determine whether acquired skewing is a consequence of stem cell depletion, true clonal hematopoiesis, growth advantage conferred by parental-specific X-chromosomes, or other causes. These data provide an explanation for variation in reported incidence of excessive skewing in normal females. Furthermore, these findings suggest that any study of clonality using X-inactivation based assays should incorporate age-matched controls for Lyonization.

Retrovirus-mediated gene transfer was used to study the effects of dysregulated expression of the zinc-finger transcription factor, GATA-1, which has been shown to be required for erythropoiesis. A retroviral vector (PGK-GATA-1) was constructed with the murine GATA-1 gene linked to the human phosphoglycerate kinase (PGK) promoter. Expression of GATA-1 was demonstrated by super-shift analysis with a monoclonal antibody against murine GATA-1 using extracts of nonerythroid cytotoxic T-lymphocyte line (CTLL) cells transduced with the PGK-GATA-1 virus. Mouse bone marrow cells were transduced in vitro and transplanted into recipient animals. Polymerase chain reaction (PCR) analysis performed on DNA extracted from peripheral blood 12 to 40 weeks posttransplantation demonstrated the presence of the PGK-GATA-1 provirus. Proviral integrity and copy number were demonstrated by Southern blot analysis of DNA from spleen, thymus, and bone marrow tissues from the long-term animals. At 16 weeks posttransplant, animals that received cells transduced by the GATA-1 virus maintained a lower white blood cell (WBC) count and absolute neutrophil count (ANC) and a higher red blood cell (RBC) count than control animals that received cells transduced with a virus containing a neor gene. Erythropoiesis was stimulated in GATA-1 and control animals by phlebotomy. GATA-1 animals required more extensive phlebotomy to reach a hematocrit less than 25 and their hematocrit returned to normal levels sooner than control animals. The effect of twice-daily injections of 10 U recombinant erythropoietin (epo) was also examined. The hematocrit of GATA-1 animals showed a more rapid and elevated response to epo than the hematocrit of control animals. These data suggest that dysregulated expression of GATA-1 in primitive hematopoietic cells enlarges the pool of epo-responsive erythroid progenitor cells.

We evaluated the methylation status of the X-linked gene phosphoglycerate kinase (PGK1) and the DXS 255 locus detected by probe M27 beta to study clonality in acquired aplastic anemia (AA). A total of 30 females were suitable for clonal analysis of peripheral blood polymorphonuclear cells (PMN) and mononuclear cells using a polymerase chain reaction-based procedure in 24 patients and Southern blotting in 9. Overall, 10 of 30 patients exhibited an imbalanced X-inactivation pattern. However, in 4 patients, analysis of constitutional DNA suggested a skewed methylation pattern and 2 further cases had to be excluded because of the lack of an appropriate control. A truly clonal pattern was thus established in 4 of 30 (13%) patients. In 7 patients who later developed clonal disorders of hematopoiesis, X-inactivation analysis did not predict this event in any case. In patients with a paroxysmal nocturnal hemoglobinuria phenotype, there was no correlation between the proportion of phosphatidylinositol glycan anchored protein (PIG-AP)-deficient blood cells and the corresponding X-inactivation pattern. X-inactivation analysis detected clonal hematopoiesis in only 3 of 10 patients with a deficiency in PIG-AP in the cell population under study, but sorting of nucleated cells on the basis of PIG-AP expression showed the clonal nature of PIG-AP-deficient cells. We conclude that the majority of patients with AA show polyclonal hematopoiesis using X-linked clonal analysis, but that minor clonal populations, such as PIG-AP-deficient cells, may not be detected unless sorted cell populations are separately analyzed.

The hierarchical level of stem cell involvement in acute promyelocytic leukemia (APL) characterized by the pathognomonic PML-RARA fusion gene is unknown. To determine if the cells of the primitive hematopoietic stem cell compartment are involved in the leukemic process, we have used molecular and cell sorting techniques in peripheral blood and bone marrow (BM) cells at diagnosis from three patients with APL and t(15; 17). In two of them, clonality analysis was also possible using the BstXI polymorphic site of the PGK gene. The PML-RARA fusion gene was readily identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of BM cells obtained at diagnosis in all three patients. These same samples were then used to sort CD34+ cells and their CD38+ and CD38- subsets by fluorescence-activated cell sorting. In both female patients, CD34+/CD38+ and CD34+/CD38- cell fractions were polyclonal using PCR, whereas a monoclonal pattern was identified at the BM sample obtained at diagnosis either by Southern blotting or by PCR. Because of the high sensitivity of the PCR analysis, the polyclonal pattern of these cell populations could mask the presence of a minor clone. To detect this clone, we preformed RT-PCR analysis for t(15; 17). In one female patient, the abnormal PML-RAR fusion gene was found only in the more mature CD34+/CD38+ cell fraction using a nested PCR approach, whereas the polyclonal CD34+/CD38- fraction was PML-RARA negative. These findings were confirmed in a third patient with APL in whom the PML-RARA transcripts were absent in the CD34+/CD38- cell fraction. To study the clonality at the level of clonogenic progenitors, we used in one patient PGK analysis by PCR of individual burst-forming units-erythroid and colony-forming units-granulocyte-macrophage obtained from the CD34+/CD38- and CD34+/CD38+ cell populations at diagnosis and from the BM sample obtained during remission. The two highly purified cell populations gave rise to morphologically normal colonies clonal for both the BstXI site containing (A) and the BstXI site lacking (B) PGK allelles, indicating their polyclonal content, a pattern that was also found in clonogenic progenitors obtained at remission. These findings strongly suggest that the primitive hematopoietic stem cells as defined by the CD34+/CD38- antigens are not involved by the neoplastic process in APL. These results may have important implications for autografting strategies of retinoic acid/chemotherapy-resistant or relapsed patients.

Juvenile chronic myelogenous leukemia (JCML) is a myeloproliferative disease in which morbidity and mortality are primarily caused by nonhematopoietic organ failure from myelomonocytic infiltration or by failure of the normal bone marrow. Morphologic evidence of maturation arrest, karyotypic abnormalities, and progression to blast crisis are infrequent events. Viral infections and other reactive processes can initially mimic the clinical course of JCML, creating diagnostic problems. Because of the rarity of JCML and technical limitations, formal clonality studies have not been reported previously. Nine female JCML patients were identified by clinical criteria, characteristic 'spontaneous' in vitro cell growth, and negative cultures and titers for various viral agents. Peripheral blood and bone marrow samples were obtained at the time of diagnosis for cell separation and RNA and DNA isolation. To assess clonality, X-chromosome inactivation patterns were evaluated using three different, recently developed polymerase chain reaction-based clonality assays. All nine female JCML patients showed evidence for monoclonal origin of mononuclear cells at the time of diagnosis. Cell separation studies further traced the monoclonal origin back to at least the most primitive myeloid progenitor cell. Reversion to a polyclonal state was demonstrated after bone marrow transplant and also in one patient following treatment with 13-cis retinoic acid. This demonstration of clonality in JCML delineates it from the reactive processes and provides a basis for molecular genetic strategies to identify causally associated mutations.

Phosphoglycerate kinase (PGK) deficiency is generally associated with chronic hemolytic anemia, although it can be accompanied by either mental retardation or muscular disease. Genomic DNAs of two PGK-deficient patients previously described in France were sequenced directly after polymerase chain reaction amplification. The PGK Créteil variant arises from a G-->A nucleotide interchange at position 1022 in cDNA (exon 9), resulting in amino acid substitution 314 Asp-->Asn in the C-terminal domain, which contains the nucleotide binding site. It is associated with rhabdomyolysis crises but not with hemolysis or mental retardation. In the other case, which is associated with chronic hemolytic anemia and mental retardation (PGK Amiens), an A-->T nucleotide interchange was found at position 571 in cDNA (exon 5); this leads to amino acid substitution 163 Asp-->Val in the N-terminal domain, which contains the catalytic site for phosphoglycerate binding. These results corroborate the kinetic data observed. In the two cases, the mutations are distinct from others previously reported and no significant relationship could be observed between the location of the amino acid substitution and its clinical consequences.

Retroviral mediated gene transfer into stem cells has been proposed as therapy for many inherited hematopoietic diseases. Deficiency of the enzyme adenosine deaminase (ADA) results in depletion of T lymphocytes, causing severe combined immunodeficiency syndrome (SCIDS). In this report, we describe retroviral mediated gene transfer of a murine ADA cDNA into Rhesus monkey hematopoietic stem cells. Immunoselected CD34+ bone marrow cells were exposed to medium containing the ADA retrovirus during culture on a stromal cell line engineered to express the transmembrane form of stem cell factor. After infusion of autologous, transduced cells into irradiated recipients, gene transfer was observed in all three monkeys. The ADA provirus was detected in 2% of circulating granulocytes and T cells from 100 days post-transplantation to longer than 1 year and in B cells from 250 days post-transplantation and beyond. Mouse ADA activity was detected in peripheral blood cells at approximately 3% the activity of monkey ADA. Thus, we have shown gene transfer into repopulating cells that contribute to all hematopoietic lineages with persistent gene expression. These data provide support for the use of stem cell targeted gene transfer for therapy of ADA deficiency.

The lymphoproliferative disease of granular lymphocytes (LDGL) results from chronic proliferation of either CD3- or CD3+ large granular lymphocytes (LGLs). Most CD3+ LGLs have been well characterized as clonal LGL proliferations (LGL leukemia). In contrast, the clonal nature and clinical features of patients with CD3- LDGL have not been defined. In this study, we analyzed seven female patients with CD3- LDGL who were heterozygous at certain X-linked gene loci. Neutrophils and CD3- granular lymphocytes were isolated from peripheral blood. Clonal analysis was performed on genomic DNA from these cell fractions on six patients by conventional Southern techniques using probes to the X-linked genes, PGK and DXS255 (M27 beta). In four patients, three of whom were already studied by Southern analyses of genomic DNA and one in whom there were insufficient amounts of DNA, polymerase chain reaction (PCR)-based clonal analysis was performed with primer pairs flanking the BstXI polymorphism on the PGK gene. In six patients, a polyclonal expansion of CD3- granular lymphocytes was demonstrated and in one the result was indeterminate. In contrast to patients with CD3+ LDGL (LGL leukemia), a clonal disease could not be demonstrated with X-linked markers in patients with CD3- LDGL, suggesting a reactive rather than a neoplastic origin of the lymphocytes in these cases.

Interstitial deletions of the long arm of chromosome 5 are among the most characteristic abnormalities observed in myeloid disorders. To assess the lineage involvement of peripheral blood cells from patients with a 5q--anomaly, purified neutrophils, monocytes, T lymphocytes, and B lymphocytes were analyzed for loss of heterozygosity using six different highly polymorphic mininucleotide and dinucleotide (CA) repeat sequences from the 5q31 to 5q33 region. Ten patients were screened by polymerase chain reaction (PCR) amplification and proved to be informative for at least one marker. Six patients showed a complete or partial disappearance of an allele in myeloid cells, whereas cells of lymphoid lineages exhibited full heterozygosity. The other patients displayed no allelic loss, indicating that the informative markers were located outside the deleted chromosomal segments. In addition, three female patients who were also polymorphic for the BstXI site in the PGK-1 gene were analyzed for the methylation status of this gene. Clonality of hematopoiesis, as determined by non-random X-chromosome inactivation, followed the same cell pattern as the 5q-specific allelic losses. In conclusion, using tumor-specific and clonal markers, we have demonstrated that the 5q- anomaly is restricted to cells of myeloid origin, leaving lymphoid cells unaffected.

To determine the clonal nature of hematopoiesis and to assess lineage involvement in patients with myelodysplastic syndromes (MDS), we used restriction fragment length polymorphisms of the X-linked genes phosphoglycerate kinase (PGK1) and hypoxanthine phosphoribosyltransferase (HPRT) and the X-linked probe M27 beta. Eleven female MDS patients heterozygous for at least one of these probes were studied: 3 with refractory anemia (RA), 2 with RA with ringed sideroblasts (RARS), 2 with chronic myelomonocytic leukemia (CMML), and 4 with RA with excess of blasts in transformation (RAEB-t). All exhibited clonal hematopoiesis as determined by Southern analysis of DNA prepared from peripheral blood (PB) and/or bone marrow (BM) cells. In three of the six patients heterozygous for the PGK1 gene, purified cell suspensions of polymorphonuclear cells (PMN), monocytes, lymphocytes, and/or T cells prepared from PB were tested. In addition, five of these patients were analyzed by a polymerase chain reaction (PCR)-based procedure as described recently. This method was slightly adapted to facilitate the analysis of cell lysates of fluorescence-activated cell sorted (FACS) monocytes, T and B lymphocytes, and natural killer (NK) cells. The outcome of Southern and PCR analysis was concordant, showing that PMN and monocytes were clonally derived, whereas circulating T and B lymphocytes and NK cells exhibited random X-chromosome inactivation compatible with a polyclonal pattern. To address the question of whether T cells are derived from unaffected progenitor cells or that their origin had antedated the onset of MDS, naive and memory T cells were analyzed separately. Both subsets showed a polyclonal pattern. However, in one patient analysis of constitutive DNA suggested a skewed methylation, and the presence of clonal lymphocytes against a background of polyclonal lymphoid cells cannot be ruled out in this patient. PCR analysis of PB and BM cells showed a nonrandom, unilateral pattern of X-inactivation, compatible with a mixture of clonally (myeloid) and polyclonally (lymphoid) derived cells. In conclusion, in some patients, MDS represents a disorder with clonal hematopoiesis restricted to cells of myeloid origin, whereas a random X-inactivation pattern is found in lymphoid cells.

A new case of X chromosome-linked phosphoglycerate kinase (PGK) abnormality is described. The male proband was mentally retarded, had behavior disorders, and displayed episodes of hemolytic anemia. The enzyme activity of red blood cells from the patient was about 10% of normal, and that of the cultured fibroblasts was about 50% of normal cells. The variant PGK was characterized by a lower affinity for the substrates, reduced thermostability, and increased anodal electrophoretic mobility. The pH activity profile of the variant enzyme was different from that of normal. The amount of messenger RNA (mRNA) in the variant fibroblasts was comparable to that of normal fibroblasts. The mRNA coding for PGK was subjected to coupled reverse transcription followed by amplification by the polymerase chain reaction. Nucleotide sequence of the variant cDNA showed a point mutation, T/A----C/G transition, in exon 9 of the variant gene. No other mutation was found in all coding regions of the variant. The mutation should cause Cys----Arg substitution at the 315th position from the NH2-terminal Ser of PGK, and it created an additional Ava II (or isoschimatic) cleavage site in the variant gene. Because the variant gene was not detected in the proband's mother and siblings, it must have been generated by spontaneous mutation during oogenesis.

We have determined a single amino acid substitution in a new phosphoglycerate kinase (PGK) variant, PGK Shizuoka, associated with chronic hemolysis and myoglobinuria. PGK Shizuoka had an extremely low enzyme activity with normal kinetic properties and normal electrophoretic mobility. Total blood cell RNA of the patient was reverse-transcribed and amplified by the polymerase chain reaction. A single nucleotide substitution from guanine to thymine at position 473 of PGK messenger RNA was found. This nucleotide change causes a single amino acid substitution from Gly to Val at the 157th position, which is located in the NH2-terminal domain of the enzyme. This mutation creates a new Bst XI cleavage site in exon 5, and we thus confirmed the mutation in the variant gene. The replacement of Gly by Val is considered to affect enzyme catalysis.

To determine whether patients with acquired asplastic anemia (AA) exhibit clonal hematopoiesis, we used restriction fragment length polymorphisms of the X-linked genes phosphoglycerate kinase (PGK1) and hypoxanthine phosphoribosyltransferase (HPRT) and the X-linked probe M27 beta. Of the 19 female patients studied, 18 (95%) patients were informative for at least one marker. Of these, eight patients (42%) were heterozygous for PGK1, two (11%) for HPRT, and 16 (84%) for M27 beta. In 13 (72%) patients, a monoclonal pattern was found. Analysis of purified cell suspensions of four of these patients showed that both myeloid and lymphoid cells were of monoclonal origin, indicating the involvement of an early stem cell. The four patients who were studied at presentation all showed a monoclonal pattern. One of these patients showed a spontaneous recovery despite persistent clonal hematopoiesis. The presence of either clonal or polyclonal hematopoiesis did not show a correlation with the response to antithymocyte globulin (ATG) treatment. A relapse after ATG was also seen in a patient exhibiting polyclonal hematopoiesis. Conversely, a monoclonal pattern did not preclude the occurrence of a partial or complete response to ATG. Other potential markers to study clonality, including cytogenetic abnormalities or point mutations of the N-ras protooncogene, were not found in any of the patients. It is concluded that patients with AA may exhibit clonal hematopoiesis. The significance with respect to evolution to disorders with clonal hematopoiesis like paroxysmal nocturnal hemoglobinuria, myelodysplasia, and acute leukemia remains to be determined.

We have identified the mutation in a phosphoglycerate kinase variant (PGK-Matsue) associated with severe enzyme deficiency, congenital nonspherocytic hemolytic anemia, and mental disorders. The mRNA coding for PGK was reverse transcribed and amplified by the polymerase chain reaction. Nucleotide sequencing of the variant cDNA showed a point mutation, a T/A----C/G transition in exon 3 of the variant gene. No other mutation was found in all coding regions of PGK-Matsue. The nucleotide change created an additional NciI cleavage site in the variant gene; thus, the NciI fragment types detected by Southern blot hybridization differ in the variant DNA and normal DNA. The mutation should cause Leu----Pro substitution at the 88th position from the NH2-terminal Ser of PGK. Because the Leu----Pro substitution is expected to induce serious perturbation and instability in the protein structure, the severe enzyme deficiency is mainly caused by more rapid in vivo denaturation and degradation of the variant enzyme.

Neonates with Down's syndrome occasionally show an excess of blasts in their peripheral blood. This disorder spontaneously resolves within several months and is called transient abnormal myelopoiesis (TAM) or transient myeloproliferative disorder. It has been uncertain whether the excess of blasts in TAM is a result of a clonal proliferation or a polyclonal reactive condition. The clonality of cells in females can be examined by analysis of the methylation patterns of the X chromosomes of proliferating cells using restriction fragment length polymorphism (RFLP). Using this strategy, we studied three females with Down's syndrome accompanied by TAM who showed heterozygosity in RFLP of either the hypoxanthine phosphoribosyltransferase or phosphoglycerate kinase gene. Analysis of the methylation patterns of these genes demonstrated a clonal nature for blasts in three patients. Thus, TAM is a clonal proliferative disorder. In addition, lymphocytes with a normal appearance contained in analyzed samples from these patients also showed a monoclonal pattern, suggesting that TAM may be a disorder of multipotent stem cells.

We used the X-linked restriction fragment length polymorphism (RFLP)-methylation strategy to study the clonal basis of the myelodysplastic syndrome (MDS) in seven patients. RFLP-methylation analysis was performed on cell populations from bone marrow (BM) aspirates and peripheral blood using probes specific for the hypoxanthine phosphoribosyltransferase (HPRT) or phosphoglycerate kinase (PGK) gene regions. Density gradient centrifugation methods were used to separate granulocytes and monocytes, and T lymphocytes were positively selected by CD2 (a pan-T marker) immunoconjugated magnetic beads. Cell populations from BM aspirates in 6 of the 7 patients with MDS showed a monoclonal pattern of X-inactivation. The neutrophilic and T-lymphocytic cell fractions were analyzed in 4 of the 6 patients, and the monocytic cell fraction in one of these, and all fractions analyzed showed a similar monoclonal pattern. In 2 of the latter 4 patients, both of whom had normal karyotypes, DNA from a skin biopsy showed a polyclonal pattern. Our data suggest that MDS is a clonal disorder, even in the absence of detectable cytogenetic abnormalities, and that the abnormal clone is capable of myeloid, monocytic, and lymphoid differentiation.

We investigated whether the stem cell that reconstitutes total erythropoiesis of a WBB6F1-W/Wv mouse differentiates into lymphoid lineage. The electrophoretic pattern of hemoglobin was used as a marker of the reconstitution; 3-phosphoglycerate kinase (PGK), an X chromosome-linked enzyme was used as a tool for estimating clonality. We injected 10(5) bone marrow cells of 5-FU treated C57BL/6-Pgk-1b/Pgk-1a female mice, in which each stem cell had either A-type PGK or B-type PGK due to random inactivation of one of two X chromosomes, into genetically anemic (WB x C57BL/6)F1-W/Wv (hereafter WBB6F1-W/Wv) mice that contained only B-type PGK. The recipient WBB6F1-W/Wv mice, in which erythropoiesis was reconstituted with donor cells for a long term, were killed and the PGK patterns of bone marrows, thymus, lymph nodes, and Peyer's patches were examined. A considerable amount of A-type PGK was detected in the lymphoid organs of the WBB6F1-W/Wv mice in which erythrocytes showed only A-type PGK when killed. In contrast, A-type PGK was scarcely detectable in the lymphoid organs of the WBB6F1-W/Wv mice in which erythrocytes showed only B-type PGK when killed. The present results suggest that the hematopoietic stem cells estimated by the erythropoiesis reconstituting assay differentiate into lymphoid lineage and that the long-term erythropoiesis reconstitution assay is useful for detecting the true primitive hematopoietic stem cells.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease characterized by immunodeficiency and severe thrombocytopenia in affected males, but no demonstrable clinical abnormalities in carrier females. Through analysis of the methylation patterns of X-linked genes that display restriction fragment length polymorphisms (RFLPs), we studied the pattern of X-chromosome inactivation in various cell populations from female relatives of patients with WAS. The peripheral blood T cells, granulocytes, and B cells of eight obligate WAS carriers were found to display specific patterns of X-chromosome inactivation clearly different from these of normal controls. Thus, carriers of WAS could be accurately identified using this analysis.