Mcph1, mutated in primary microcephaly, is also crucial for erythropoiesis.

Microcephaly is a common feature in inherited bone marrow failure syndromes, prompting investigations into shared pathways between neurogenesis and hematopoiesis. To understand this association, we studied the role of the microcephaly gene Mcph1 in hematological development. Our research revealed that Mcph1-knockout mice exhibited congenital macrocytic anemia due to impaired terminal erythroid differentiation during fetal development. Anemia's cause is a failure to complete cell division, evident from tetraploid erythroid progenitors with DNA content exceeding 4n. Gene expression profiling demonstrated activation of the p53 pathway in Mcph1-deficient erythroid precursors, leading to overexpression of Cdkn1a/p21, a major mediator of p53-dependent cell cycle arrest. Surprisingly, fetal brain analysis revealed hypertrophied binucleated neuroprogenitors overexpressing p21 in Mcph1-knockout mice, indicating a shared pathophysiological mechanism underlying both erythroid and neurological defects. However, inactivating p53 in Mcph1-/- mice failed to reverse anemia and microcephaly, suggesting that p53 activation in Mcph1-deficient cells resulted from their proliferation defect rather than causing it. These findings shed new light on Mcph1's function in fetal hematopoietic development, emphasizing the impact of disrupted cell division on neurogenesis and erythropoiesis - a common limiting pathway.

Autogenous Control of 5'TOP mRNA Stability by 40S Ribosomes.

Ribosomal protein (RP) expression in higher eukaryotes is regulated translationally through the 5'TOP sequence. This mechanism evolved to more rapidly produce RPs on demand in different tissues. Here we show that 40S ribosomes, in a complex with the mRNA binding protein LARP1, selectively stabilize 5'TOP mRNAs, with disruption of this complex leading to induction of the impaired ribosome biogenesis checkpoint (IRBC) and p53 stabilization. The importance of this mechanism is underscored in 5q− syndrome, a macrocytic anemia caused by a large monoallelic deletion, which we found to also encompass the LARP1 gene. Critically, depletion of LARP1 alone in human adult CD34+ bone marrow precursor cells leads to a reduction in 5'TOP mRNAs and the induction of p53. These studies identify a 40S ribosome function independent of those in translation that, with LARP1, mediates the autogenous control of 5'TOP mRNA stability, whose disruption is implicated in the pathophysiology of 5q− syndrome.

Macrocytic anemias.

PURPOSE OF REVIEW: Over the last century, the diseases associated with macrocytic anemia have been changing with more patients currently having hematological diseases including malignancies and myelodysplastic syndrome. The intracellular mechanisms underlying the development of anemia with macrocytosis can help in understanding normal erythropoiesis. Adaptations to these diseases involving erythroid progenitor and precursor cells lead to production of fewer but larger red blood cells, and understanding these mechanisms can provide information for possible treatments. RECENT FINDINGS: Both inherited and acquired bone marrow diseases involving primarily impaired or delayed erythroid cell division or secondary adaptions to basic erythroid cellular deficits that results in prolonged cell division frequently present with macrocytic anemia. SUMMARY OF FINDINGS: In marrow failure diseases, large accumulations of iron and heme in early stages of erythroid differentiation make cells in those stages especially susceptible to death, but the erythroid cells that can survive the early stages of terminal differentiation yield fewer but larger erythrocytes that are recognized clinically as macrocytic anemia. Other disorders that limit deoxynucleosides required for DNA synthesis affect a broader range of erythropoietic cells, but they also lead to macrocytic anemia. The source of macrocytosis in other diseases remains uncertain.

Hematologic presentation and the role of untargeted metabolomics analysis in monitoring treatment for riboflavin transporter deficiency.

Riboflavin transporter deficiency (RTD) (MIM #614707) is a neurogenetic disorder with its most common manifestations including sensorineural hearing loss, peripheral neuropathy, respiratory insufficiency, and bulbar palsy. Here, we present a 2-year-old boy whose initial presentation was severe macrocytic anemia necessitating multiple blood transfusions and intermittent neutropenia; he subsequently developed ataxia and dysarthria. Trio-exome sequencing detected compound heterozygous variants in SLC52A2 that were classified as pathogenic and a variant of uncertain significance. Bone marrow evaluation demonstrated megaloblastic changes. Notably, his anemia and neutropenia resolved after treatment with oral riboflavin, thus expanding the clinical phenotype of this disorder. We reiterate the importance of starting riboflavin supplementation in a young child who presents with macrocytic anemia and neurological features while awaiting biochemical and genetic work up. We detected multiple biochemical abnormalities with the help of untargeted metabolomics analysis associated with abnormal flavin adenine nucleotide function which normalized after treatment, emphasizing the reversible pathomechanisms involved in this disorder. The utility of untargeted metabolomics analysis to monitor the effects of riboflavin supplementation in RTD has not been previously reported.

The impact of preoperative anaemia and anaemic subtype on patient outcome in colorectal cancer.

AIM: Preoperative anaemia is associated with adverse outcomes in colorectal cancer (CRC). To clarify the reason for this we aimed to comprehensively assess the association of preoperative anaemia with tumour characteristics, host systemic inflammation and nutrition status, and perioperative blood transfusion. METHOD: We used an integrated database of 592 CRC patients. The association of preoperative anaemic subtype, calculated from haemoglobin and erythrocyte mean corpuscular volume levels, with patient outcome, preoperative serum data relating to systemic inflammation and nutrition and perioperative blood transfusion was analysed. RESULTS: Preoperative anaemia was significantly associated with poorer overall survival and relapse-free survival (RFS); in particular microcytic anaemia had a trend to poorer RFS than other forms of anaemia (P = 0.0648). In addition, preoperative anaemia was significantly correlated with right-sided tumours, greater depth of tumour invasion, use of neoadjuvant chemotherapy, poorer prognostic nutritional index and higher modified Glasgow Prognostic Score (mGPS). Microcytic anaemia in particular had a strong association with a greater depth of tumour invasion (P = 0.0072) and higher mGPS (P = 0.0058) than other causes of anaemia. Perioperative blood transfusion for CRC patients with anaemia was associated with adverse outcomes. CONCLUSIONS: Preoperative anaemia, especially microcytic anaemia, was associated with poor patient outcomes, possibly due to poor systemic inflammatory and nutritional status, and it was not improved by perioperative blood transfusion. Our data suggest that preoperative anaemia and the anaemic subtype may serve as an easily available predictor of outcome in CRC.

Bone marrow features in Pearson syndrome with neonatal onset: A case report and review of the literature.

Pearson syndrome (PS) is a rare mitochondrial disorder that usually presents with transfusion-dependent macrocytic anemia, exocrine pancreatic dysfunction, and lactic acidosis. Typical bone marrow (BM) features are vacuolization in hematopoietic progenitors, hypocellularity, and ringed sideroblasts. At the neonatal age, PS may have a variable clinical onset. Moreover, there is little information about BM features at this age and the timing of their presentation. We report a neonatal case of PS that presented with refractory anemia and atypical BM features. We reviewed the BM findings in neonatal-onset PS cases to stress the importance and limitations of BM evaluation at this age.

Dyserythropoiesis of myelodysplastic syndromes.

PURPOSE OF REVIEW: Myelodysplastic syndromes (MDS) are heterogeneous diseases of the hematopoietic stem cell in the elderly. Anemia is the main symptom that mostly correlates with dysplastic erythropoiesis in the bone marrow. We will review the recent advances in understanding the diverse mechanisms of dyserythropoiesis. RECENT FINDINGS: Dyserythropoiesis defined as 10% dysplastic erythroid cells in the bone marrow is found in more than 80% of early MDS. Immature erythroblasts accumulate at the expense of mature erythroblasts due to differentiation arrest and apoptosis. In early MDS with dyserythropoiesis, caspase-dependent cleavage of the erythroid transcription factor GATA-1 occurring in basophilic erythroblasts accounts for impairment of final maturation. Depending on initiating genetic alteration, specific mechanisms contribute to erythroid defect. In MDS with 5q deletion, the haploinsufficiency of ribosomal protein gene, RPS14, opposes the transition of immature to mature erythroblasts by inducing a p53-dependent ribosome stress, cell cycle arrest and apoptosis. Recent work identifies the activation of a p53-S100A8/9 innate immune pathway that both intrinsically and extrinsically contributes to defective erythropoiesis. In MDS with ring sideroblasts, a paradigm of dyserythropoiesis, a unique mutation in SF3B1 splicing factor gene induces a multiplicity of alterations at RNA level that deeply modifies the patterns of gene expression. SUMMARY: Insights in the pathophysiology of MDS with dyserythropoiesis may guide the choice of the appropriate therapy, for instance lenalidomide in MDS with del(5q). A better understanding of the mechanisms of dyserthropoiesis is required to treat anemia in non-del(5q) MDS, especially in case of resistance to first-line therapy by erythropoiesis-stimulating agents.

High level of full-length cereblon mRNA in lower risk myelodysplastic syndrome with isolated 5q deletion is implicated in the efficacy of lenalidomide.

Downregulation of cereblon (CRBN) gene expression is associated with resistance to the immunomodulatory drug lenalidomide and poor survival outcomes in multiple myeloma (MM) patients. However, the importance of CRBN gene expression in patients with myelodysplastic syndrome (MDS) and its impact on lenalidomide therapy are not clear. In this study, we evaluate cereblon expression in mononuclear cells isolated from bone marrow [23 lower risk MDS patients with isolated 5q deletion (5q-), 37 lower risk MDS patients with chromosome 5 without the deletion of long arms (non-5q-), and 24 healthy controls] and from peripheral blood (38 patients with 5q-, 52 non-5q- patients and 25 healthy controls) to gain insight into, firstly, the role of cereblon in lower risk MDS patients with or without 5q deletion and, secondly, into the mechanisms of lenalidomide action. Patients with 5q- lower risk MDS have the highest levels of CRBN mRNA in comparison with both lower risk MDS without the deletion of long arms of chromosome 5 and healthy controls. CRBN gene expression was measured using the quantitative TaqMan real-time PCR. High levels of CRBN mRNA were detected in all lenalidomide responders during the course of therapy. A significant decrease of the CRBN mRNA level during lenalidomide treatment is associated with loss of response to treatment and disease progression. These results suggest that, similar to the treatment of MM, high levels of full-length CRBN mRNA in lower risk 5q- patients are necessary for the efficacy of lenalidomide.

Genome-wide miRNA profiling in myelodysplastic syndrome with del(5q) treated with lenalidomide.

OBJECTIVES: Lenalidomide is a potent drug with pleiotropic effects in patients with myelodysplastic syndrome (MDS) with deletion of the long arm of chromosome 5 [del(5q)]. We investigated its effect on regulation of microRNA (miRNA) expression profiles in del(5q) patients with MDS in vivo. METHODS: We used miRNA expression microarrays to study changes in miRNA levels in peripheral blood CD14+ monocytes collected from patients before and during lenalidomide treatment and compared them with those from healthy donors. RESULTS: Before treatment, we observed strong upregulation of pro-apoptotic miR-34a and miR-34a* that diminished during lenalidomide exposure. Upregulation of HOX-related miR-196b and erythroid-specific miR-451 seen in untreated patients remained unchanged after the treatment. At the time of hematologic response, expression of several miRNAs clustering to the 14q32 locus was reduced. Additionally, we focused more deeply on miRNAs from the 5q commonly deleted region and found that levels of miR-378 and miR-378* followed haploinsufficiency trend. CONCLUSIONS: This report describes changes in miRNA expression in del(5q) patients with MDS treated with lenalidomide, likely arising from deregulation of pathways implicated in lenalidomide action.

L-Leucine improves the anaemia in models of Diamond Blackfan anaemia and the 5q- syndrome in a TP53-independent way.

Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q- myelodysplastic syndrome. We previously demonstrated that treatment with L-Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L-Leucine effect was Tp53-dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L-leucine treatment. We confirmed this observation in human CD34+ cells. L-Leucine thus alleviates anaemia in RP-deficient cells in a TP53-independent manner.

L-Leucine improves the anemia and developmental defects associated with Diamond-Blackfan anemia and del(5q) MDS by activating the mTOR pathway.

Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34⁺ cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS.

Nonclinical Safety Profile of BMS-986001, a Nucleoside Transcriptase Inhibitor for Combination Retroviral Therapy.

Nucleoside reverse transcriptase inhibitors (NRTIs)/nucleotide reverse transcriptase inhibitors are key components of combination antiretroviral therapy for HIV infection. First-generation NRTIs are associated with mitochondrial toxicity in patients, mainly due to inhibition of human DNA polymerase γ (hDNA polγ) that manifests as adverse events such as lipodystrophy, lactic acidosis, myopathy, cardiomyopathy, or nephropathy in patients. In chronic nonclinical studies in rodents and nonrodents, eukaryotic (host) mitochondrial toxicity manifests as some drug-specific toxicities similar to human toxicity. BMS-986001, a novel thymidine analog with minimal hDNA polγ inhibition, has demonstrated antiretroviral activity in early clinical studies. The primary toxicity of BMS-986001 in rats and monkeys is bone marrow dyserythropoiesis with associated decreases in red blood cell mass. Additionally, at high doses, severe platelet reductions accompanied by cutaneous petechiae began during weeks 8 and 11 in 3 of 60 monkeys in chronic toxicity studies. In a 6-month study, platelet reductions required euthanasia of the 2 affected monkeys (300 mg/kg/d) at week 14, but with dose reduction (200 mg/kg/d) remaining monkeys had no platelet changes. One affected monkey (200 mg/kg/d) in a 9-month study completed dosing and its platelet counts recovered during a 1-month recovery. Formation of platelet-bound immunoglobulin in the presence of BMS-986001, together with rapid and complete platelet recovery in the absence of BMS-986001, suggested that platelet decreases in monkeys may be immune mediated. No findings indicative of mitochondrial toxicity were observed in rats or monkeys given BMS-986001, suggesting an improved safety profile compared to marketed NRTI or tenofovir disoproxil fumarate.

Transcription factors Fli1 and EKLF in the differentiation of megakaryocytic and erythroid progenitor in 5q- syndrome and in Diamond-Blackfan anemia.

Friend leukemia virus integration 1 (Fli1) and erythroid Krüppel-like factor (EKLF) participate under experimental conditions in the differentiation of megakaryocytic and erythroid progenitor in cooperation with other transcription factors, cytokines, cytokine receptors, and microRNAs. Defective erythropoiesis with refractory anemia and effective megakaryopoiesis with normal or increased platelet count is typical for 5q- syndrome. We decided to evaluate the roles of EKLF and Fli1 in the pathogenesis of this syndrome and of another ribosomopathy, Diamond-Blackfan anemia (DBA). Fli1 and EKLF mRNA levels were examined in mononuclear blood and bone marrow cells from patients with 5q- syndrome, low-risk MDS patients with normal chromosome 5, DBA patients, and healthy controls. In 5q- syndrome, high Fli1 mRNA levels in the blood and bone marrow mononuclear cells were found. In DBA, Fli1 expression did not differ from the controls. EKLF mRNA level was significantly decreased in the blood and bone marrow of 5q- syndrome and in all DBA patients. We propose that the elevated Fli1 in 5q- syndrome protects megakaryocytic cells from ribosomal stress contrary to erythroid cells and contributes to effective though dysplastic megakaryopoiesis.

Haploinsufficiency of Apc leads to ineffective hematopoiesis.

Loss of a whole chromosome 5 or a deletion of the long arm of chromosome 5, -5/del(5q), is a recurring abnormality in myeloid neoplasms. The APC gene is located at chromosome band 5q23, and is deleted in more than 95% of patients with a -5/del(5q), raising the question of whether haploinsufficiency of APC contributes to the development of myeloid neoplasms with loss of 5q. We show that conditional inactivation of a single allele of Apc in mice leads to the development of severe anemia with macrocytosis and monocytosis. Further characterization of the erythroid lineage revealed that erythropoiesis is blocked at the early stages of differentiation. The long-term hematopoietic stem cell (LT-HSC) and short-term HSC (ST-HSC) populations are expanded in Apc-heterozygous mice compared with the control littermates; however, the HSCs have a reduced capacity to regenerate hematopoiesis in vivo in the absence of a single allele of Apc. Apc heterozygous myeloid progenitor cells display an increased frequency of apoptosis, and decreased in vitro colony-forming capacity, recapitulating several characteristic features of myeloid neoplasms with a -5/del(5q). Our results indicate that haploinsufficiency of Apc impairs hematopoiesis, and raise the possibility that loss of function of APC contributes to the development of myelodysplasia.

Perturbed hematopoiesis in the Tc1 mouse model of Down syndrome.

Trisomy of human chromosome 21 (Hsa21) results in Down syndrome (DS), a disorder that affects many aspects of physiology, including hematopoiesis. DS children have greatly increased rates of acute lymphoblastic leukemia and acute megakaryoblastic leukemia (AMKL); DS newborns present with transient myeloproliferative disorder (TMD), a preleukemic form of AMKL. TMD and DS-AMKL almost always carry an acquired mutation in GATA1 resulting in exclusive synthesis of a truncated protein (GATA1s), suggesting that both trisomy 21 and GATA1 mutations are required for leukemogenesis. To gain further understanding of how Hsa21 contributes to hematopoietic abnormalities, we examined the Tc1 mouse model of DS, which carries an almost complete freely segregating copy of Hsa21, and is the most complete model of DS available. We show that although Tc1 mice do not develop leukemia, they have macrocytic anemia and increased extramedullary hematopoiesis. Introduction of GATA1s into Tc1 mice resulted in a synergistic increase in megakaryopoiesis, but did not result in leukemia or a TMD-like phenotype, demonstrating that GATA1s and trisomy of approximately 80% of Hsa21 perturb megakaryopoiesis but are insufficient to induce leukemia.

Reticulocyte Hemoglobin Equivalent in Patients with Idiopathic Warm Autoimmune Hemolytic Anemia: Implication in the Development of Macrocytosis.

OBJECTIVE: Patients with warm autoimmune hemolytic anemia (WAIHA) present with anemia that is highly heterogeneous, and often have macrocytic anemia with inappropriately elevated mean corpuscular volume (MCV). The goal of this retrospectivecase study is to elucidate the characteristics of anemia in patients with idiopathic WAIHA. PROCEDURES: The hematological parameters were analyzed by automated hematology analyzers in 19 consecutive patients with idiopathic WAIHA. Thecontent of hemoglobin (Hb) in the reticulocytes was assessed as reticulocyte Hb equivalent (RET-He). Relevant laboratory data and medical records were retrospectively studied. RESULTS: The median MCV was 102.7 fL and ten patients had macrocytic anemia with MCV above 100 fL. There was a significant correlation between the percentage of reticulocytes and MCV. The median RET-He value was 35.9 pg, and the reticulocytes of patients with higher MCV had higher RET-He. There was a significant correlation between red cell volume distribution width (RDW) andMCV, while the association between RDW and RET-He was not significant. Red blood cell agglutination was not seen in any of the patients. Relative folate deficiency was implied to contribute to the increased Hb content in the reticulocytes of WAIHA patients. CONCLUSION: Reticulocytes in WAIHA patients often contain more Hb than normal reticulocytes and become inappropriately large, possibly due to relative folate deficiency. Elevated MCV in WAIHA patients is due to the increase in both the number and the Hb content of reticulocytes.

Rps14, Csnk1a1 and miRNA145/miRNA146a deficiency cooperate in the clinical phenotype and activation of the innate immune system in the 5q- syndrome.

RPS14, CSNK1A1, and miR-145 are universally co-deleted in the 5q- syndrome, but mouse models of each gene deficiency recapitulate only a subset of the composite clinical features. We analyzed the combinatorial effect of haploinsufficiency for Rps14, Csnk1a1, and miRNA-145, using mice with genetically engineered, conditional heterozygous inactivation of Rps14 and Csnk1a1 and stable knockdown of miR-145/miR-146a. Combined Rps14/Csnk1a1/miR-145/146a deficiency recapitulated the cardinal features of the 5q- syndrome, including (1) more severe anemia with faster kinetics than Rps14 haploinsufficiency alone and (2) pathognomonic megakaryocyte morphology. Macrophages, regulatory cells of erythropoiesis and the innate immune response, were significantly increased in Rps14/Csnk1a1/miR-145/146a deficient mice as well as in 5q- syndrome patient bone marrows and showed activation of the innate immune response, reflected by increased expression of S100A8, and decreased phagocytic function. We demonstrate that Rps14/Csnk1a1/miR-145 and miR-146a deficient macrophages alter the microenvironment and induce S100A8 expression in the mesenchymal stem cell niche. The increased S100A8 expression in the mesenchymal niche was confirmed in 5q- syndrome patients. These data indicate that intrinsic defects of the 5q- syndrome hematopoietic stem cell directly alter the surrounding microenvironment, which in turn affects hematopoiesis as an extrinsic mechanism.

Defective DNA synthesis in human megaloblastic bone marrow: effects of homocysteine and methionine.

In B(12) deficiency, inadequate DNA synthesis seems due in large measure to a block of tetrahydrofolic acid (THFA) regeneration from 5-methyl THFA (via homocysteine transmethylation). In support of the above, homocysteine appears to facilitate and methionine to reduce de novo DNA synthesis. This was measured by the ability of deoxyuridine to suppress thymidine-(3)H uptake into DNA in human bone marrow cultures. The homocysteine effect in B(12)-deficient marrow supports the possibility that there is in man an additional B(12)-independent pathway for regeneration of THFA by methylation of homocysteine to form methionine. Among possible explanations for the methionine effect is end-product inhibition of the homocysteine transmethylase reaction, resulting in further accumulation of 5-methyl THFA. Homocysteine transmethylation may play an important role in the regulation of THFA availability and de novo DNA synthesis. In vitro and in vivo evidence suggests that methionine may be useful to potentiate and homocysteine to reduce methotrexate action.

Altered vitamin B12 metabolism in fibroblasts from a patient with megaloblastic anemia and homocystinuria due to a new defect in methionine biosynthesis.

Cultured fibroblasts from a recently described patient with homocystinuria and megaloblastic anemia of infancy without methylmalonic aciduria were previously shown to have normal cobalamin uptake and a specific decrease in the proportion of intracellular methylcobalamin. As in control cells but unlike in those from patients with combined homocystinuria and methylmalonic aciduria (cobalamin C and cobalamin D), accumulated 57Co-labeled cobalamin was bound in appropriate amounts and proportion to intracellular binders which are known to be the two vitamin B12-dependent enzymes, methionine synthetase and methylmalonyl-CoA mutase. Despite the association of a normal quantity of intracellular cobalamin with methionine synthetase, the proportion of intracellular cobalamin which was methyl-B12 was below normal and in the range observed in cobalamin C and D cells. This methyl-B12 was decreased by exposure of fibroblasts in culture to nitrous oxide as was observed with control cells. Exposure of control fibroblasts during culture, but not of fibroblasts from this patient, to nitrous oxide significantly reduced the holoenzyme activity of methionine synthetase assayed in cell extracts. In addition, although methionine synthetase activity in cell extracts of control and cells from the patient were similar in the presence of standard assay concentrations of thiols, at low thiol concentrations, methionine synthetase activity in extracts of cells from the patient was much lower than in control extracts. Mixing of control patient extracts corrected this decreased activity in excess of that explained by addition of the individual activities added. The defect of this patient appears to be in a reducing system required for methionine synthesis.