Characterization and Genetic Studies of Microcytic Anemia in House Mouse

Microcytic anemia, inherited as a unit autosomal recessive, is the first of thetwelve known single-gene induced hereditary anemias of the mouse whichresults entirely from alteration of individual erythrocytes rather than from reduction in number of red cells. At all ages where mk/mk individuals could berecognized, from the fifteenth day of gestation to adulthood, the erythrocytecount of mk/mk individuals was at least as high as that of normal counterparts.At all ages, the hemoglobin concentration in these small erythrocytes was alsoreduced, so that the total available hemoglobin was markedly reduced at allages. The higher than normal numbers of erythrocytes in adult microcyticmice demonstrate that the cell-producing mechanisms operate efficiently.Genetic tests have shown that the mk single-gene defect has no relation tostructure of either the -chain or the -chain of the hemoglobin molecule. Themk microcytosis and hypochromia must then result from a metabolic or structural defect independent both of the factors responsible for regulating red cellnumber and of those controlling hemoglobin structure.

This mk/mk microcytic anemia shows some similarities to human thalassemias: hypochromia, microcytosis and presence of target cells, combined withsplenomegaly, reticulocytosis, and higher than normal erythrocyte counts (likethalassemia minor). As in thalassemia, hemoglobin structure is normal, although the amount per cell is subnormal. Human hemolytic anemias also sharesome of these characteristics. The similarities of mk/mk microcytic anemia ofthe mouse to certain human anemias are sufficient to warrant its further investigation as an animal model for understanding of human hereditary disease.

Submitted on October 9, 1969 Accepted on January 15, 1970     

The Nramp2/DMT1 iron transporter is induced in the duodenum of microcytic anemia mk mice but is not properly targeted to the intestinal brush border

Microcytic anemia (mk) mice and Belgrade (b) rats are severely iron deficient because of impaired intestinal iron absorption and defective iron metabolism in peripheral tissues. Both animals carry a glycine to arginine substitution at position 185 in the iron transporter known as Nramp2/DMT1 (divalent metal transporter 1). DMT1 messenger RNA (mRNA) and protein expression has been examined in the gastrointestinal tract of mk mice. Northern blot analysis indicates that, by comparison to mk/+ heterozygotes, mk/mk homozygotes show a dramatic increase in the level of DMT1 mRNA in the duodenum. This increase in RNA expression is paralleled by a concomitant increase of the 100-kd DMT1 isoform I protein expression in the duodenum. Immunohistochemical analyses show that, as for normal mice on a low-iron diet, DMT1 expression in enterocytes of mk/mk mice is restricted to the duodenum. However, and in contrast to normal enterocytes, little if any expression of DMT1 is seen at the apical membrane in mk/mk mice. These results suggest that the G185R mutation, which was shown to impair the transport properties of DMT1, also affects the membrane targeting of the protein in mk/mk enterocytes. This loss of function of DMT1 is paralleled by a dramatic increase in expression of the defective protein in mk/mk mice. This is consistent with a feedback regulation of DMT1 expression by iron stores. (Blood. 2000;96:3964-3970)     

Microcytic anemia with iron malabsorption: An inherited disorder of iron metabolism

Two siblings were identified with severe hypoproliferative microcytic anemia and iron malabsorption, in the absence of any gastrointestinal disorder or blood loss. These children had severe microcytosis (MCV 48 fl, hemoglobin 7.5 g/dl) with decreased serum iron, elevated serum TIBC, and decreased serum ferritin, despite prolonged treatment with oral iron. An iron challenge study with an oral dose of 2 mg/kg elemental iron as ferrous sulfate documented iron malabsorption. After treatment with intravenous iron dextran, there was an absence of the expected reticulocytosis and only a partial correction of the hemoglobin, hematocrit, and microcytosis. The bone marrow was hypocellular with abnormal iron incorporation into erythroid precursor cells. This appears to be a rare form of inherited anemia characterized by iron malabsorption and disordered iron metabolism that only partially corrects after the administration of parenteral iron. These features resemble those found in the microcytic mouse (mk/mk), which also has severe microcytic anemia and iron malabsorption that partially responds to parenteral iron. © 1996 Wiley-Liss, Inc.     

Nramp2 is mutated in the anemic Belgrade (b) rat: Evidence of a role for Nramp2 in endosomal iron transport

The Belgrade (b) rat has an autosomal recessively inherited, microcytic, hypochromic anemia associated with abnormal reticulocyte iron uptake and gastrointestinal iron absorption. The b reticulocyte defect appears to be failure of iron transport out of endosomes within the transferrin cycle. Aspects of this phenotype are similar to those reported for the microcytic anemia (mk) mutation in the mouse. Recently, mk has been attributed to a missense mutation in the gene encoding the putative iron transporter protein Nramp2. To investigate the possibility that Nramp2 was also mutated in the b rat, we established linkage of the phenotype to the centromeric portion of rat chromosome 7. This region exhibits synteny to the chromosomal location of Nramp2 in the mouse. A polymorphism within the rat Nramp2 gene cosegregated with the b phenotype. A glycine-to-arginine missense mutation (G185R) was present in the b Nramp2 gene, but not in the normal allele. Strikingly, this amino acid alteration is the same as that seen in the mk mouse. Functional studies of the protein encoded by the b allele of rat Nramp2 demonstrated that the mutation disrupted iron transport. These results confirm the hypothesis that Nramp2 is the protein defective in the Belgrade rat and raise the possibility that the phenotype shared by mk and b animals is unique to the G185R mutation. Furthermore, the phenotypic characteristics of these animals indicate that Nramp2 is essential both for normal intestinal iron absorption and for transport of iron out of the transferrin cycle endosome.     

Red cell iron uptake in hereditary microcytic anemia

The iron uptake in vitro of red cells from mice with hereditary microcytic anemia (gene symbol mk) was studied to examine the hypothesis of a generalized impairment of cellular iron uptake in this conidition. Reticulocyte-rich red cells from anemic (mk/mk) and acutely bled normal (+/+) mice were incubated in 59Fe-labeled mouse plasma and the radioiron uptake measured. The 59Fe uptake of the mk/mk and +/+ cells was related in the same way to the reticulocyte concentration, the duration of incubation, and the percentage saturation of the plasma iron-binding capacity. However, under the same conditions, the iron uptake of red cells from normal (+/+) mice was greater than that by red cells from anemic (mk/mk) mice. Furthermore, the cellular loss of radioiron on exposure to EDTA was greater for the mk/mk red cells, although the proportion of the radioiron taken up that was incorporated into heme was the same for mk/mk and +/+ red cells. These results support the hypothesis of a generalized impairment of cellular iron uptake in hereditary microcytic anemia and suggest that there might be a defect in red cell receptor sites for transferrin in this condition.     

The Cellular Basis of the Genetically Determined Hemopoietic Defect in Anemic Mice of Genotype Sl/Sld

The proliferation and function of hemopoietic cells derived from geneticallyanemic Sl/Sl^d mice have been studied by the use of cell transplantationtechnics. It was found that marrow cells derived from anemic Sl/Sl^d or Sl^d/Sl^dmice, when implanted into heavily irradiated mice of genotype +^sl/+^sl, arecapable of forming macroscopic spleen colonies, with approximately the samefrequency as cells derived from normal +^sl/+^sl mice. Marrow cells derivedfrom Sl/Sl^d animals were tested for their capacity to cure the anemia of W/W^rmice and were found to implant as rapidly and to have as long-lasting abeneficial effect as did marrow cells from +^sl/+^sl mice. The radiosensitivityof the colony-forming ability of marrow cells from Sl/Sl^d mice was found tobe similar to that found previously for +^sl/+^sl marrow cells, although theanemic animals are known to be more sensitive to total-body irradiation thanare their normal littermates. Marrow cells from +^sl/+^sl mice were found toproliferate more slowly in irradiated mice of genotype Sl/Sl^d than in irradiated+^sl/+^sl littermates, even when the anemic and the normal mice were joinedin parabiosis. These observations indicate that the hemopoietic colony-formingcells in mice of genotype Sl/Sl^d are normal, but fail to function adequately because the tissues of mice of this genotype are unable to provide sufficient support for proliferation and differentiation of these progenitor cells.

Submitted on November 25, 1964 Accepted on December 25, 1964     

Hematopoietic Cells From alpha -Spectrin-Deficient Mice Are Sufficient to Induce Thrombotic Events in Hematopoietically Ablated Recipients

Thrombotic events are life-threatening complications of humanhemolytic anemias such as paroxysmal nocturnal hemoglobinuria, sicklecell disease, and thalassemia. It is not clear whether these events aresolely influenced by aberrant hematopoietic cells or also involveaberrant nonhematopoietic cells. Spherocytosis mutant(Spna1^sph/Spna1^sph; for simplicityreferred to as sph/sph) mice develop a severe hemolytic anemiapostnatally due to deficiencies in -spectrin in erythroid and otheras yet incompletely defined nonerythroid tissues. Thrombotic lesionsoccur in all adult sph/sph mice, thus providing ahematopoietically stressed model in which to assess putative causes ofthrombus formation. To determine whether hematopoietic cells fromsph/sph mice are sufficient to initiate thrombi, bone marrowfrom sph/sph or +/+ mice was transplanted into mice with nohemolytic anemia. One set of recipients was lethally irradiated; theother set was genetically stem cell deficient. All mice implanted withsph/sph marrow, but not +/+ marrow, developed severe anemia and histopathology typical of sph/sph mice. Histologicalanalyses of marrow recipients showed that thrombi were present in therecipients of sph/sph marrow, but not +/+ marrow. Theresults indicate that the -spectrin-deficient hematopoietic cellsof sph/sph mice are the primary causative agents of thethrombotic events.     

Reticulocyte Survival in Sickle Cell Anemia: Effect of Cyanate

Cyanate prevents sickling in vitro andapparently prolongs the survival of⁵¹Cr-tagged sickle erythrocytes in vivo.Cautious interpretation is required because the effects of cyanate on ⁵¹Crbinding to sickle and fetal hemoglobin-containing red cells are unknown, andcomparison of the effect of cyanate onsickle red cell survival to control redcell survival must be performed sequentially. We have studied the survival of sickle reticulocytes utilizingradioactive amino acids that are incorporated into hemoglobin. Two informed adult patients with sickle celldisease were studied. In each study,two 50-ml samples of blood were incubated separately with ¹⁴C- and ³H-leucine for 2 hr, after which 50 mMcyanate was added to one aliquot for1 hr. The cells were then washed andreinfused. Frequent venous sampleswere obtained, and the specific activities of ¹⁴C and ³H in the hemoglobinwere followed. The t of the carbamylated cells was tripled, but remainedbelow normal. This method provides agenerally useful measurement of theinfluence of drugs bound to red cellson reticulocyte lifespan. The labels areincorporated into the hemoglobinmolecule of the reticulocyte, andsimultaneous comparison of the survivals of the same cohort of drug-treated and control cells is achieved.

Submitted on May 2, 1972 Revised on June 16, 1972 Accepted on June 26, 1972     

Erythrocyte Fe59 Uptake as a Function of Bone Marrow Dose Injected in Lethally Irradiated Mice

The relation between bone marrow cell dose and 24-hour erythrocyte Fe⁵⁹uptake has been established in lethally irradiated mice. Erythrocyte Fe⁵⁹ uptake is a function of the dose of bone marrow cells and of the time after irradiation at which Fe⁵⁹ is injected. By choosing appropriate bone marrowdoses and times of Fe⁵⁹ injection, the range of cell doses between 5 x 10⁵ and2 x 10⁷ has been explored. The relation between cell dose and Fe⁵⁹ uptake islinear for Fe⁵⁹ uptakes between 0 to 30 per cent. The steepest line relatingFe⁵⁹ uptake to cell dose is that obtained when Fe⁵⁹ was injected at day 9 andcovers the range of 5 X 10⁴ to 5 X 10⁵ cells. The curve obtained when ironis injected on day 5 is much flatter and covers the range of 1 x 10⁶ to 2 x10⁷ cells. Erythropoiesis stimulating factor (ESF) in doses that stimulateerythrocyte Fe⁵⁹ uptake in normal mice has no effect in irradiated, bone marrow-treated mice. Homologous marrow is slightly less effective, and rat bonemarrow markedly ( 100 times) less effective in promoting recovery of erythropoieis. The erythrocyte Fe⁵⁹ uptake of mice preimmunized with homologous or rat marrow before irradiation is much lower than that of nonpreimmunized animals.

Submitted on October 26, 1961 Accepted on January 12, 1962     

Importance of anemia and transferrin levels in the regulation of intestinal iron absorption in hypotransferrinemic mice.

The hypotransferrinemic mouse (trf (hpx)) is a mutant strain exhibiting transferrin deficiency, marked anemia, hyperabsorption of iron, and elevated hepatic iron stores. We set out to investigate the relative roles of anemia and of transferrin in the malregulation of intestinal iron absorption in these animals. Transfusion of erythrocytes obtained from littermate controls increased hemoglobin levels and reduced reticulocyte counts in recipient animals. Although mucosal to carcass (59)Fe transfer was reduced, total duodenal iron uptake was not significantly affected. Iron absorption in homozygotes, in contrast to littermate controls, was not reduced by hyperoxia. Mouse transferrin injections, in the short term, increased delivery of iron to the marrow and raised hemoglobin levels. Although mucosal transfer and total iron uptake were reduced at the higher transferrin doses, total uptake was still higher than in controls. Daily injections of mouse/human transferrin for 3 weeks from weaning, normalized hemoglobin values, and markedly reduced liver iron and intestinal iron absorption values in trf (hpx) animals. When such daily-injected mice were left for a week to allow transferrin clearance, iron absorption values were significantly enhanced; hemoglobin or hepatic iron levels were, however, not significantly altered. These data indicate that hyperabsorption of iron in trf (hpx) mice is not solely because of the anemia; transferrin levels per se do affect iron absorption, possibly via a direct effect on the intestinal mucosa.     

Glycogen Metabolism in the Normal Red Blood Cell

Evidence for active glycogen metabolism in normal mature red blood cells(RBC) is presented. Initial rates of¹⁴C-U-glucose incorporation into erythrocyte glycogen were found to beindependent of substrate concentration over a range of 3.3-16.6 mM. Incorporation of label into glycogen wasinitially linear but reached a plateauafter a variable period of time that wasinversely related to RBC concentrationin the medium. The major part of theincorporated radioactivity resided inthe outer branches of the glycogenmolecule. The optimum pH for ¹⁴C-U-glucose incorporation into glycogenwas pH 7.6. Replacing the radioactiveglucose employed for incorporationafter 1 hr of incubation with nonlabeledglucose resulted in a gradual loss ofradioactivity from erythrocyte glycogen. In normal cells, glycogen synthesis and breakdown do not result inany significant accumulation of glycogen, whereas in erythrocytes withenzyme defects affecting glycogenbreakdown, substantial deposition ofglycogen may be observed.

Submitted on March 10, 1972 Revised on July 7, 1972 Accepted on July 10, 1972     

Prediction of iron deficiency in chronic inflammatory rheumatic disease anaemia

Summary. We prospectively studied 45 anaemic patients (3 7 women, 8 men) with chronic inflammatory rheumatic diseases. The combination of serum ferritin and CRP (as well as ESR) in its predictive capacity for bone marrow iron stores was examined. The relationship between other iron-related measurements (transferrin, transferrin saturation, soluble transferrin receptor, erythrocyte porphyrins and percentage of hypochromic/microcytic erythrocytes) and bone marrow iron stores was also investigated. Stainable bone marrow iron was taken as the most suitable standard to separate iron-deficient from iron-replete patients. 14 patients (31%) were lacking bone marrow iron. Regression analysis showed a good correlation between ferritin and bone marrow iron (adjusted R ²=0.721, P<00001). The combination of ferritin and CRP (ESR) did not improve the predictive power for bone marrow iron (adjusted R ²=0.715) in this cohort of patients with low systemic inflammatory activity. With respect to the bone marrow iron content the best predictive cut-off value of ferritin was 30μg/l (86% sensitivity, 90% specificity). The other iron-related parameters both individually and when combined were less powerful in predicting bone marrow iron than ferritin alone. Only zinc bound erythrocyte protoporphyrin in combination with ferritin slightly improved prediction (adjusted R ²=0.731). A cut-off point of 11% hypochromic erythrocytes reached a high specificity (90%), but was less sensitive (77%).     

RAP‐011, an activin receptor ligand trap,increases hemoglobin concentration in hepcidin transgenic mice

Over expression of hepcidin antimicrobial peptide is a common feature of iron‐restricted anemia in humans. We investigated the erythroid response to either erythropoietin or RAP‐011, a “murinized” ortholog of sotatercept, in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. Sotatercept, a soluble, activin receptor type IIA ligand trap, is currently being evaluated for the treatment of anemias associated with chronic renal disease, myelodysplastic syndrome, β‐thalassemia, and Diamond Blackfan anemia and acts by inhibiting signaling downstream of activin and other Transforming Growth Factor‐β superfamily members. We found that erythropoietin and RAP‐011 increased hemoglobin concentration in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. While erythropoietin treatment depleted splenic iron stores in C57BL/6 mice, RAP‐011 treatment did not deplete splenic iron stores in mice of either genotype. Bone marrow erythroid progenitors from erythropoietin‐treated mice exhibited iron‐restricted erythropoiesis, as indicated by increased median fluorescence intensity of transferrin receptor immunostaining by flow cytometry. In contrast, RAP‐011‐treated mice did not exhibit the same degree of iron‐restricted erythropoiesis. In conclusion, we have demonstrated that RAP‐011 can improve hemoglobin concentration in hepcidin antimicrobial peptide 1 transgenic mice. Our data support the hypothesis that RAP‐011 has unique biologic effects which prevent or circumvent depletion of mouse splenic iron stores. RAP‐011 may, therefore, be an appropriate therapeutic for trials in human anemias characterized by increased expression of hepcidin antimicrobial peptide and iron‐restricted erythropoiesis. Am. J. Hematol. 90:8–14, 2015. © 2014 Wiley Periodicals, Inc.     

Iron deficiency anaemia in hospitalised patients: value of various laboratory parameters. Differentiation between IDA and ACD.

BACKGROUND: for the diagnostic evaluation of microcytic or normocytic anaemia in a heterogeneous group of patients, the value of newer parameters, such as zinc protoporphyrin (ZPP), plasma transferrin receptor (PtrfR) and PtrfR/ferritin ratio is not clear. We have performed a prospective study to determine the predictive value of these parameters and ferritin, for diagnosing iron deficiency anaemia (IDA). METHODS: sixty-two patients with Hb<8.2 (men) or <7.0 (women) and mean cell volume (MCV)<96 fl were included. Exclusion criteria were: known haematological disease, pregnancy, bone marrow suppression or iron therapy within the previous 7 days. Bone marrow examination was used as a golden standard to discriminate between IDA and non-IDA. RESULTS: twenty-four patients had depleted iron stores. We found that the reticulocyte response on iron supplementation correlated well with the iron-status of the bone marrow. Univariate analysis showed that ferritin, PtrfR/ferritin ratio, ZPP and PtrfR have significant predictive values for differentiating IDA from non-IDA. Interestingly, multivariate analysis revealed that ferritin was the only significant, independent predictor of IDA, with a cut-off point of 32 microg/l (sensitivity 79.2%, specificity 96.9%). CONCLUSIONS: the low sensitivity and specificity of ZPP, PtrfR and PtrfR/ferritin ratio render them insufficient to be used as a single 'best' test for the identification IDA in a non-selected group of anaemic patients and do not even add to the prediction if the value of ferritin is known.     

Erythropoiesis in Carotid Body Resected Cats

It has been reported that removal of carotid bodies in cats results in a brief, profuse reticulocytosis followed by depressionto below-normal levels of reticulocytesand a progressively severe anemia. Injection of cat carotid body extract was reported to increase erythrocyte ⁵⁹Fe incorporation in polycythemic rats. Otherstudies could find no hematological abnormality in humans after bilateral carotidbody resection. We reexamined the effectof carotid body resection in the cat withserial bone marrow aspirations, hematocrit determination, and reticulocyte determination. T_1/2 for plasma ⁵⁹Fe removal,erythrocyte ⁵⁹Fe incorporation, and ⁵¹Crlifespan determinations were performedon five operated and three control cats.No significant differences were found. Weconclude that the carotid body has nodirect effect on erythropoiesis and thatthe anemia reported in a prior study wassecondary to sepsis resulting from an indwelling femoral vein catheter.

Submitted on February 15, 1973 Revised on April 20, 1973 Accepted on April 25, 1973     

Leukocyte Interferon Production, RNA Synthesis, and PHA Response in Patients With Infectious Mononucleosis

Sequential measurements of in vitro Newcastle disease virus-induced interferon(IF) synthesis, spontaneous RNA synthesis, and PHA-induced blastic transformation were made in leukocytes derived fromten patients with infectious mononucleosis (IM). The median leukocyte IF production on the day of diagnosis was foundto be significantly depressed, 35 international units (IU) IF per 2 x 10⁶ leukocytes,in comparison to a median titer of 463 IUafter recovery (p < 0.002) or to a medianof 406 IU found in 52 cultures from normalhealthy individuals. No spontaneous production of leukocyte IF or circulating serumIF was noted. An increased rate of spontaneous leukocyte RNA synthesis duringthe first 2 hr of culture was noted in nineof the ten subjects (p < 0.001). Four ofthe ten patients showed a markedly defective response to PHA after 96-144 hrin culture. A suggestive, but not statistically significant, correlation was notedbetween increased spontaneous RNA synthesis, defective late PHA transformation,and defective virus-induced IF production.All defects persisted during the 1-wk studyperiod and were resolved after recovery.We conclude that major temporary alterations occur in several parameters of leukocyte function in IM.

Submitted on September 11, 1972 Revised on December 18, 1972 Accepted on January 15, 1973     

Regeneration of Marrow Tissue in Chronic Iron Deficiency in Postweanling Rats

The regenerative potential of chronically iron-deficient rat marrow wasstudied in extramedullary marrowautotransplants and intramedullarycavity after ablation. Subcutaneousimplantation of normal marrow resultsconsistently in establishment of a marrow nodule with a mean weight ratioof 30.7% of the implanted tissue. Iniron deficiency, the mean weight ratiowas 7.1%, and the take was 11%.After ablation of femoral marrow, theregenerative process proceeds distallyfrom the uninjured marrow at thefemoral head. The process of regeneration reached the midshaft after 11days in iron-deficient animals but tookonly 5 days for normal animals. Ironrepletion accelerated the process toward normal. These results are consistent with biochemical data fromiron-deficient marrow (low nucleic acidcontent, decreased incorporation of³H-thymidine into DNA, and decreasedutilization of ⁵⁹Fe and ¹⁴C-glycine forheme synthesis) and suggest that thereported hypercellularity of the marrowin iron deficiency may reflect sequestration of erythroid precursors, ratherthan the compensatory mechanism ofincreased cell proliferation.

Submitted on July 28, 1971 Revised on January 24, 1972 Accepted on January 29, 1972     

Immunologic Identification of Tissue Factor (Thromboplastin) Synthesized by Cultured Fibroblasts

The proliferative capacity of bone marrowcells from thymus-deprived nude mice wasinvestigated in lethally irradiated recipients. Although the colony-forming capacityof these cells was found to be similar tothat of normal littermates, a reduction inthe number of nucleated cells was observed in the bone marrow of nude mice.Moreover when the radioprotective effectof such cells was studied, it was foundthat 5 x 10⁵ or 2 x 10⁶ bone marrowcells from nude mice were less effectivein restoring hemopoiesis and establishingpermanent chimerism than similaramounts of bone marrow cells of normalcontrols. In addition, irradiated animalssurviving after injection of bone marrowcells from nude mice were found to havelower immune responses to SRBC thannormal chimeras. The possibility that mortality of irradiated recipients injected withbone marrow of nude mice is due to thepresence of a latent infective agent or ofsome inhibitory factor of hematopoiesis inthe bone marrow of such nude mice isshown to be improbable. Alternatively it issuggested that nude mice suffer from anintrinsic defect in the proliferative capacityof their bone marrow colony-forming cells(CFUs).

Submitted on February 12, 1973 Revised on March 29, 1973 Accepted on April 20, 1973     

Characterization of primitive hematopoietic cells from patients with dyskeratosis congenita

Dyskeratosis congenita (DC) is an inherited bone marrow (BM) failure syndrome associated with mutations in telomerase genes and the acquisition of shortened telomeres in blood cells. To investigate the basis of the compromised hematopoiesis seen in DC, we analyzed cells from granulocyte colony-stimulating factor mobilized peripheral blood (mPB) collections from 5 members of a family with autosomal dominant DC with a hTERC mutation. Premobilization BM samples were hypocellular, and percentages of CD34⁺ cells in marrow and mPB collections were significantly below values for age-matched controls in 4 DC subjects. Directly clonogenic cells, although present at normal frequencies within the CD34⁺ subset, were therefore absolutely decreased. In contrast, even the frequency of long-term culture-initiating cells within the CD34⁺ DC mPB cells was decreased, and the telomere lengths of these cells were also markedly reduced. Nevertheless, the different lineages of mature cells were produced in normal numbers in vitro. These results suggest that marrow failure in DC is caused by a reduction in the ability of hematopoietic stem cells to sustain their numbers due to telomere impairment rather than a qualitative defect in their commitment to specific lineages or in the ability of their lineage-restricted progeny to execute normal differentiation programs.