Biallelic Variants in the Ectonucleotidase ENTPD1 Cause a Complex Neurodevelopmental Disorder with Intellectual Disability, Distinct White Matter Abnormalities, and Spastic Paraplegia.

OBJECTIVE: Human genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683). METHODS: Individuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed. RESULTS: A total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414-2_414-1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574-6_574-3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574-6_574-3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism. INTERPRETATION: The ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1-related neurological disease, (2) highlights the importance of genotype-driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022;92:304-321.

The JAK2V617F mutation in normal individuals takes place in differentiating cells.

The JAK2V617F mutation that results in a hyper-activation of the JAK2 kinase in the erythropoietin pathway is a molecular marker for myeloproliferative neoplasms. Using allele-specific Real-Time PCR, we detected the mutation in the blood of 17.3% (17/98) of normal donors; the mutant allele burden was, however, very low (<0.01% compared to >1% in polycythemia vera). It was much higher in differentiated blood cells in the peripheral blood than in undifferentiated CD34+ cells. Erythropoietin-stimulated differentiation of normal CD34+ cells in liquid culture increased the mutation frequency by 3.34-fold. When progenitors from 9 normal donors were grown in erythropoietin-stimulated semi-solid cultures, the mutation was found in 8.69% of the colonies, but only in <3% of the JAK2 alleles in each positive colony, suggesting that the mutation occurred only in a few cells per colony. In mouse erythroleukemia cells carrying human JAK2 DNA, wild-type or JAK2V617F, the frequencies of mutations from JAK2 wild-type to JAK2V617F and vice versa increased following erythroid differentiation. These results suggest that the mutation occurs and accumulates during differentiation. We hypothesize that genetic stability, which relies on DNA repair, is efficient in normal hematopoietic stem cells but is downgraded in differentiating cells, rendering them susceptible to mutations, including JAK2V617F.

Erythroid differentiation ability of butyric acid analogues: identification of basal chemical structures of new inducers of foetal haemoglobin.

Several investigations have demonstrated a mild clinical status in patients with ß-globin disorders and congenital high persistence of foetal haemoglobin. This can be mimicked by a pharmacological increase of foetal γ-globin genes expression and foetal haemoglobin production. Our goal was to apply a multistep assay including few screening methods (benzidine staining, RT-PCR and HPLC analyses) and erythroid cellular model systems (the K562 cell line and erythroid precursors collected from peripheral blood) to select erythroid differentiation agents with foetal haemoglobin inducing potential. With this methodology, we have identified a butyric acid derivative, namely the 4174 cyclopropanecarboxylic acid compound, able to induce erythroid differentiation without antiproliferative effect in K562 cells and increase of γ-globin gene expression in erythroid precursor cells. The results are relevant for pharmacological treatments of haemoglobinopathies, including ß-thalassaemia and sickle cell anaemia.

Heterogeneity of F cells in ß-thalassemia.

BACKGROUND: Fetal hemoglobin (HbF), which is largely replaced after birth by the adult Hb, is concentrated in a few "F cells." Their number significantly increases in certain physiologic and clinical situations, including in ß-thalassemia (ß-thal). Their quantification is used to detect fetal-maternal hemorrhage (FMH), where fetal cells enter the maternal circulation. We were confronted with a pregnant woman with ß-thal who was suspected to have FMH. To establish the usefulness of a flow cytometric procedure to differentiate between fetal cells and the maternal F cells, we screened adult ß-thal patients. STUDY DESIGN AND METHODS: Blood samples were simultaneously stained with fluorescent antibodies to HbF and to carbonic anhydrase (CA) isotype II, which is specific to adult red blood cells (RBCs). RESULTS: A heterogeneous distribution of RBCs with respect to HbF and CA expression was observed: adult non-F cells (CA+HbF-) and F cells (CA+HbF+/HbF++) as well as F cells with characteristics of fetal cells (CA-HbF++). CONCLUSIONS: The presence of CA-HbF++ RBCs in nonpregnant women, and even men, with thal indicates that the CA/HbF method is inappropriate for detection of FMH. The coexistence of F cells carrying fetal or adult markers suggests that they originate from two types of stem cell, adult and fetal, lineages. Normally, the fetal lineage is insignificant, but in ß-thal, as HbF-containing RBCs have a selective advantage, the "fetal" lineage gains significance.

Thalassemic DNA-Containing Red Blood Cells Are under Oxidative Stress.

We studied the nature of enucleated RBCs containing DNA remnants, Howell-Jolly (HJ) RBCs and reticulocytes (retics), that are characteristically present in the circulation of thalassemic patients, especially after splenectomy. Using flow cytometry methodology, we measured oxidative status parameters of these cells in patients with ß-thalassemia. In each patient studied, these cells had higher content of reactive oxygen species and exposed phosphatidylserine compared with their DNA-free counterparts. These results suggest that oxidative stress in thalassemic developing erythroid precursors might, through DNA-breakage, generate HJ-retics and HJ-RBCs and that oxidative stress-induced externalization of phosphatidylserine is involved in the removal of these cells from the circulation by the spleen, a mechanism similar to that of the removal of senescent RBCs.

Therapeutic hemoglobin levels after gene transfer in ß-thalassemia mice and in hematopoietic cells of ß-thalassemia and sickle cells disease patients.

Preclinical and clinical studies demonstrate the feasibility of treating ß-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human ß-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients.We generated lentiviral vectors carrying the human ß-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human ß-globin through a novel mechanism that links the rate of transcription of the transgenic ß-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34(+) cells isolated from patients affected by ß-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S).Our results suggest two major findings. First, we discovered that for the purpose of expressing the ß-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from ß-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.

Resveratrol: Antioxidant activity and induction of fetal hemoglobin in erythroid cells from normal donors and ß-thalassemia patients.

Thalassemia and sickle-cell anemia (SCA) present a major public health problem in countries where the number of carriers and affected individuals is high. As a result of the abnormalities in hemoglobin production, cells of thalassemia and SCA patients exhibit oxidative stress, which ultimately is responsible for the chronic anemia observed. Therefore, identification of compounds exhibiting both antioxidant and hemoglobin-inducing activities is highly needed. Our results demonstrate resveratrol to be such a compound. This was shown both in the human K562 cell line, as well as in erythroid precursors derived from normal donors and ß-thalassemia patients. Resveratrol was shown to exhibit antioxidant activity and to stimulate the expression of the γ-globin genes and the accumulation of fetal hemoglobin (HbF). To the best of our knowledge, this is the first report pointing to such a double effect of resveratrol. Since this natural product is already marketed as an antioxidant, future investigations should concentrate on demonstrating its potential to augment HbF production in experimental animal models (e.g., thalassemia and SCA mice) as well as in patients. We believe that the potential of clinical use of resveratrol as an antioxidant and HbF stimulator may offer a simple and inexpensive treatment to patients.

Uptake of non-transferrin iron by erythroid cells.

Most of the iron in the plasma is bound to transferrin (Tf) and is taken up by cells through their surface Tf receptors (TfRs). Under pathological conditions of iron-overload, the plasma iron which is in excess of the binding capacity of Tf is present as non-Tf-bound iron. We probed the uptake of non-Tf iron and its consequences on the oxidative status of peripheral RBC and reticulocytes as well as developing erythroid precursors grown in vitro. The cells were exposed to ferrous ammonium sulfate under Tf-supplemented and Tf-free conditions. Using flow cytometry techniques, we found that both the TfR-deficient mature RBC and their TfR-containing precursors at all stages of maturation can take up non-Tf iron that accumulates as redox-active labile iron and generates reactive oxygen species. Such a mechanism may account for ineffective erythropoiesis of developing precursors in the bone marrow and for the shortening of the lifespan of mature RBCs in the circulation.

Effect of iron chelators on labile iron and oxidative status of thalassaemic erythroid cells.

BACKGROUND/AIMS: Iron accumulation in vital organs such as heart and liver is a major pathology in beta-thalassaemia. It may also affect mature RBCs and developing erythroid precursors. The cellular damage is mainly caused by the labile iron pool (LIP) and is mediated by reactive oxygen species (ROS). We have previously shown that thalassaemic RBCs and their precursors have more LIP and ROS than their normal counterparts. We now report the effect of clinically relevant iron chelators on these parameters. METHODS: RBCs, reticulocytes and cultured erythroid precursors derived from patients with beta-thalassaemia were studied for LIP and oxidative stress parameters by flow-cytometry. RESULTS: In vitro treatment with deferiprone, deferasirox and deferoxamine reduced the cytosolic LIP in RBCs and reticulocytes, and both the cytosolic and mitochondrial LIP in cultured erythroid precursors. This was associated with reduced oxidative stress (ROS and external phosphatidylserine). While the effect of deferiprone and deferasirox was fast (within 10 min), deferoxamine affected these parameters after 24 h, suggesting a slower rate of entry. CONCLUSION: The chelators studied reduce the LIP and the oxidative status of thalassaemic RBC and their precursors. Whether these effects directly improve ineffective erythropoiesis and RBC survival remains to be shown.

Bis-epoxyethyl derivatives of distamycin A modified on the amidino moiety: induction of production of fetal hemoglobin in human erythroid precursor cells.

Derivatives of distamycin A modified at the C-terminal amidine moiety and tethered to bis-epoxyethyl moieties at the N-terminal position were tested for their ability to induce erythroid differentiation in the human erythroleukemic cell line K562. None of the compounds without bis-epoxyethyl moiety were active. A comparison of the biological activity of diepoxy compounds containing different non-basic amidine-modified moieties, showed low activity of amidoxime, carbamoyl and N-methyl carbamoyl derivatives as differentiation agents. In contrast, a cyanamidine derivative, compound 3, was able to induce erythroid differentiation of K562 cells. In addition, the cyanamidine derivative 3 was able to induce HbF accumulation following treatment of cultures of erythroid precursor cells isolated from the peripheral blood of normal subjects.

The labile iron pool in human erythroid cells.

Although most cellular iron is firmly bound (e.g. in haemoglobin), some, the labile iron pool (LIP), is bound to low-affinity ligands. The LIP is regarded as the crossroads of cellular iron traffic. Using multi-parameter flow-cytometry of cells treated with the metal-sensitive sensor calcein and the cell-permeable chelator deferiprone, we studied LIP in various human erythroid cell populations in peripheral blood, bone marrow and culture. Erythroid maturation was found to be associated with a decrease in the LIP. In the peripheral blood, nucleated erythrocytes (normoblasts) had 5.8-fold and 8.8-fold greater LIP than reticulocytes and erythrocytes respectively. Early reticulocytes had 2.5-fold more LIP than late reticulocytes. In the bone-marrow and in culture, LIP decreased by c. 30-fold as early erythroid precursors matured to late precursors. Adding holo-transferrin to iron-depleted cultures elevated LIP by 3.9-fold. We also show that in beta-thalassaemia, a disease associated with iron-overload, erythrocytes and reticulocytes in the blood and erythroid precursors in culture have a significantly greater LIP than their normal counterparts. In conclusion, the LIP in erythroid cells is altered under physiological (maturation) and pathological (thalassaemia) conditions. The methodology presented might be useful for evaluating the LIP in various diseases and for studying the efficacy of iron-chelators.

Iron-chelator complexes as iron sources for early developing human erythroid precursors.

Developing erythroid cells are dependent on transferrin (Tf) to acquire iron in amounts sufficient for hemoglobin production. Previously, we showed that although these cells cannot grow in culture in the absence of Tf, ferritin (Ft) can substitute Tf to some extent and support the development of hemoglobin-containing cells. In the current study, we investigated the ability of various iron sources to replace Tf in cultures of normal human erythroid precursors. The results showed that whereas Ft and hemin supported erythroid cell proliferation and hemoglobinization in Tf-free cultures to some extent, ferric amonium citrate and iron complexed with several chelators had little or no effect. Although salicylaldehyde-isonicotinoyl-hydrazone, which is a tridentate lipid-soluble chelator, complexed with iron increased both cytosolic and mitochondrial labile iron pools, it failed to support heme synthesis and did not decrease the surface Tf receptors, suggesting that its iron is not recognized by the cells. Moreover, this iron-chelator complex did not support erythroid precursor proliferation and hemoglobinization. Thus, although under normal conditions, Tf is the major route of iron uptake, Ft and hemin, but not iron-chelator complexes, may serve as alternative iron sources under Tf-poor conditions.

Flow cytometry measurement of the labile iron pool in human hematopoietic cells.

Iron is important for many biological processes, and its deficiency or excess is involved in pathological conditions. Although most iron is firmly bound (e.g., in hemoglobin), some, the labile iron pool (LIP), is bound to low-affinity ligands. The level of LIP is regulated to meet the cell's requirements for iron but prevent excess. We describe herein a multiparameter flow cytometry procedure for measuring LIP in various human hematopoietic cells. Peripheral blood and bone marrow (BM) cells were loaded with calcein-AM, washed, and then incubated with or without the high-affinity iron-chelator Deferiprone (L1). Specific cell subpopulations were identified based on side-light scattering and expression of surface antigens. LIP was determined based on the ability of L1 to bind and remove iron from calcein and thereby increase the fluorescence emitted by the cells. Blood cells differ in their LIP content in the order monocytes > PMN > RBC > lymphocytes. Analysis of BM cells indicated a similar tendency among precursors of the different lineages. The results also showed that among myeloid precursors, LIP increases along cell maturation. Flow cytometry might be useful for evaluating LIP in various diseases and for studying the efficacy of iron-chelators.

Macrophages function as a ferritin iron source for cultured human erythroid precursors.

Iron is essential for the survival as well as the proliferation and maturation of developing erythroid precursors (EP) into hemoglobin-containing red blood cells. The transferrin-transferrin receptor pathway is the main route for erythroid iron uptake. Using a two-phase culture system, we have previously shown that placental ferritin as well as macrophages derived from peripheral blood monocytes could partially replace transferrin and support EP growth in a transferrin-free medium. We now demonstrate that in the absence of transferrin, ferritin synthesized and secreted by macrophages can serve as an iron source for EP. Macrophages trigger an increase in both the cytosolic and the mitochondrial labile iron pools, in heme and in hemoglobin synthesis, along with a decrease in surface transferrin receptors. Inhibiting macrophage exocytosis, binding extracellular ferritin with specific antibodies, inhibiting EP receptor-mediated endocytosis or acidification of EP lysosomes, all resulted in a decreased EP growth when co-cultured with macrophages under transferrin-free conditions. The results suggest that iron taken up by macrophages is incorporated mainly into their ferritin, which is subsequently secreted by exocytosis. Nearby EP are able to take up this ferritin probably through clathrin-dependent, receptor-mediated endocytosis into endosomes, which following acidification and proteolysis release the iron from the ferritin, making it available for regulatory and synthetic purposes. Thus, macrophages support EP development under transferrin-free conditions by delivering essential iron in the form of metabolizable ferritin.

The effect of the copper chelator tetraethylenepentamine on reactive oxygen species generation by human hematopoietic progenitor cells.

Clinical observations suggest that copper (Cu) plays a role in regulating hematopoietic progenitor cell (HPC) development. Cu is known to generate oxidative stress in cells which in turn affects proliferation, differentiation and apoptosis. To study this role of Cu, we used double staining flow cytometry to measure reactive oxygen species (ROS) generation by neonatal cord blood-derived CD34(+)CD38(-) cells. ROS was increased by Cu and was decreased by the Cu chelator tetraethylenepentamine (TEPA). Previously, we showed that TEPA reduces the free Cu content of HPCs and stimulates their ex vivo expansion. The present results suggest that TEPA affects expansion of HPCs by lowering their oxidative stress.

Vanadate elevates fetal hemoglobin in human erythroid precursors by inhibiting cell maturation.

Increased fetal hemoglobin (HbF) in erythroid precursors of patients with beta-hemoglobinopathies (sickle cell anemia and beta-thalassemia), in which adult hemoglobin synthesis is defective, ameliorates the clinical symptoms of the underlying diseases. The production of erythroid precursors depends on the action of erythropoietin (EPO), which prevents their apoptosis and stimulates their proliferation. EPO binds to its surface receptor, induces its homodimerization, and initiates a cascade of phosphorylation and dephosphorylation of a series of proteins by kinases and phosphatases, respectively. Vanadate inhibits various phosphatases, including those that are involved in the EPO pathway, thereby intensifying the signal. In this study, we investigated the effect of vanadate on the proliferation and maturation of human erythroid precursors in culture. When vanadate was added to cells derived from normal donors, cell maturation was delayed, as indicated by cell morphology, cell growth kinetics, the rate of appearance of hemoglobin-containing cells, and the expression of surface antigens (CD117, CD71, and glycophorin A). Analysis by high-performance liquid chromatography and flow cytometry of the hemoglobin profile of vanadate-treated normal cells revealed a higher proportion of HbF than was found in untreated cells. When vanadate was added to cells derived from patients with beta-thalassemia, a significant increase in HbF was observed. The results suggest that intensification of the EPO signal by vanadate results in maturation arrest and increased HbF production. Thus, inhibitors that are more specific and less toxic than vanadate may present a novel option for elevating HbF in patients with beta-hemoglobinopathies, as well as for intensifying the EPO response in other forms of anemia.

Effects of rapamycin on accumulation of alpha-, beta- and gamma-globin mRNAs in erythroid precursor cells from beta-thalassaemia patients.

We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 beta-thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two-phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the beta-thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to gamma-globin mRNA accumulation, being only minor for beta-globin and none for alpha-globin mRNAs. The ability of rapamycin to preferentially increase gamma-globin mRNA content and production of HbF in erythroid precursor cells from beta-thalassaemia patients is of great importance as this agent (also known as sirolimus or rapamune) is already in clinical use as an anti-rejection agent following kidney transplantation. These data suggest that rapamycin warrants further evaluation as a potential therapeutic drug in beta-thalassaemia and sickle cell anaemia.

Chelatable cellular copper modulates differentiation and self-renewal of cord blood-derived hematopoietic progenitor cells.

OBJECTIVES: We have demonstrated epigenetic modulation of CD34(+) cell differentiation by the high-affinity copper (Cu) chelator tetraethylenepentamine (TEPA). TEPA slowed down the rate of CD34(+) cell differentiation and increased their engraftability in SCID mice. TEPA biological activity was attributed to its effect on cellular Cu levels as (a) treatment with TEPA resulted in reduction of cellular Cu, and (b) excess of Cu reversed TEPA's activity and accelerated differentiation. In the present study we further evaluated the role of cellular Cu in TEPA's biological activity. METHODS: The effects of Cu-chloride, TEPA, TEPA/Cu mixtures at various ratios, and a synthesized, stable, TEPA-Cu complex on short- and long-term cord blood-derived CD34(+) cell cultures as well as on the overall and chelatable cellular Cu were investigated. RESULTS: Addition of TEPA, TEPA/Cu mixtures at up to equimolar concentrations, and the TEPA-Cu complex to CD34(+) cell cultures resulted in inhibition of differentiation and enhancement of long-term self-renewal. Measurement of the overall cellular Cu by atomic absorption spectrophotometry showed 20 to 40% decrease by TEPA while the TEPA-Cu mixture and the TEPA-Cu complex increased cellular Cu by 10- to 20-fold, as did CuCl(2). However, measurement of the cellular pool of labile Cu showed similar reduction (50% from the control) by all the TEPA forms, while CuCl(2) increased it. Thus, inhibition of differentiation and enhancement of self-renewal of CD34(+) cells was correlated with reduction in the cellular chelatable Cu content. CONCLUSION: The results suggest that decreasing of the chelatable Cu pool, rather than overall Cu, is the mechanism that stands behind TEPA's biological activity.

Retinoic acid receptor antagonist inhibits CD38 antigen expression on human hematopoietic cells in vitro.

The CD34+ CD38- subset of human hematopoietic stem cells are crucial for long-term ex-vivo expansion; conditions that decreased this specific sub-population reduced the self-renewal capacity and shortened the duration of the proliferative phase of the culture. Retinoids, such as all-trans retinoic acid (ATRA), have been shown to induce CD38 expression. ATRA present in serum may be responsible for the high CD38 of cells grown in serum-containing medium. In the present study we analyzed the effects of AGN 194310, a retinoic acid receptor pan-antagonist, on CD38 expression of human hematopoietic cells. Normal cells (cord blood derived CD34+ cells) and abnormal cells (myeloid leukemic lines) were studied when grown in either serum-containing or serum-free media. The results showed that both serum and ATRA enhanced differentiation and, thereby, reduced the proportion of CD34+ CD38- cells and total CD34+ cell expansion. AGN reversed these effects of serum and ATRA: it delayed differentiation and increased CD34+ CD38- cells. These results suggest that physiological ATRA levels in serum may prevent efficient cell expansion. AGN, by neutralizing ATRA, improves cell expansion in serum-containing cultures, thus making AGN a useful agent for ex vivo expansion of stem cells and other specific sub-populations for research and clinical use.

Rapamycin-mediated induction of gamma-globin mRNA accumulation in human erythroid cells.

The present study aimed to determine whether rapamycin could increase the expression of gamma-globin genes in human erythroid cells. Rapamycin is a macrocyclic lactone that possesses immunosuppressive, antifungal and anti-tumour properties. This molecule is approved as an immunosuppressive agent for preventing rejection in patients receiving organ transplantation. To verify the activity of rapamycin, we employed two experimental cell systems, the human leukaemia K562 cell line and the two-phase liquid culture of human erythroid progenitors isolated from normal donors and patients with beta-thalassaemia. The results suggested that rapamycin, when compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and gamma-globin mRNA accumulation in human leukaemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of rapamycin, gamma-globin mRNA accumulation and fetal haemoglobin (HbF) production increased to levels that were higher than those obtained using hydroxyurea. These effects were not associated with inhibition of cell growth. Furthermore, rapamycin was found to increase HbF content in erythroid precursor cells from four beta-thalassaemia patients. These results could have practical relevance, because pharmacologically mediated regulation of the expression of human gamma-globin genes, leading to increased HbF, is considered a potential therapeutic approach in haematological disorders, including beta-thalassaemia and sickle cell anaemia.