Hb Switching Research Articles

Molecular and haematological characterisation of haemolytic anaemia associated with biallelic KLF1 mutations: a case series

AimsKrüppel-like factor 1 (KLF1) is an erythroid-specific transcription factor playing an important role in erythropoiesis and haemoglobin (Hb) switching. Biallelic KLF1 mutations can cause haemolytic anaemia with thalassaemia-like syndromes but...

Evaluating sheep hemoglobins with MD simulations as an animal model for sickle cell disease

Sickle cell disease (SCD) affects millions worldwide, yet there are few therapeutic options. To develop effective treatments, preclinical models that recapitulate human physiology and SCD pathophysiology are needed. SCD arises from a single Glu-to-Val substitution at position 6 in the β subunit of hemoglobin (Hb), promoting Hb polymerization and subsequent disease. Sheep share important physiological and developmental characteristics with humans, including the same developmental pattern of fetal to adult Hb switching. Herein, we investigated whether introducing the SCD mutation into the sheep β-globin locus would recapitulate SCD’s complex pathophysiology by generating high quality SWISS-MODEL sheep Hb structures and performing MD simulations of normal/sickle human (huHbA/huHbS) and sheep (shHbB/shHbS) Hb, establishing how accurately shHbS mimics huHbS behavior. shHbS, like huHbS, remained stable with low RMSD, while huHbA and shHbB had higher and fluctuating RMSD. shHbB and shHbS also behaved identically to huHbA and huHbS with respect to β2-Glu6 and β1-Asp73 (β1-Asn72 in sheep) solvent interactions. These data demonstrate that introducing the single SCD-causing Glu-to-Val substitution into sheep β-globin causes alterations consistent with the Hb polymerization that drives RBC sickling, supporting the development of a SCD sheep model to pave the way for alternative cures for this debilitating, globally impactful disease.

Pharmacological Induction of Fetal Hemoglobin in β-Thalassemia and Sickle Cell Disease: An Updated Perspective.

A significant amount of attention has recently been devoted to the mechanisms involved in hemoglobin (Hb) switching, as it has previously been established that the induction of fetal hemoglobin (HbF) production in significant amounts can reduce the severity of the clinical course in diseases such as β-thalassemia and sickle cell disease (SCD). While the induction of HbF using lentiviral and genome-editing strategies has been made possible, they present limitations. Meanwhile, progress in the use of pharmacologic agents for HbF induction and the identification of novel HbF-inducing strategies has been made possible as a result of a better understanding of γ-globin regulation. In this review, we will provide an update on all current pharmacological inducer agents of HbF in β-thalassemia and SCD in addition to the ongoing research into other novel, and potentially therapeutic, HbF-inducing agents.

Downregulation of KLF4 activates embryonic and fetal globin mRNA expression in human erythroid progenitor cells.

The Krüppel-like factor (KLF) family dominates highly conserved three zinc finger DNA binding domains at the C-terminus and variable transactivation domains at the N-terminus. Humans possess 18 KLF genes that are differentially expressed in various tissues. Several KLFs recognize a specific CACCC DNA motif that is commonly found within hematopoietic-specific promoters. To investigate those KLFs that are involved in human hemoglobin (Hb) switching, the present study analyzed a previous microarray data set from fetal and adult erythroid cells and validated the mRNA expression levels of 18 KLFs by reverse transcription-quantitative PCR (RT-qPCR). KLF with a decreased expression level in the fetuses was selected for a functional study in human erythroid progenitor cells using lentiviral-based short hairpin RNA knockdown. The fetuses demonstrated a lower level of KLF4 mRNA expression when compared with the adults. Downregulation of KLF4 in erythroid progenitor cells from healthy individuals and individuals with β0-thalassemia/HbE evidenced the increasing embryonic and fetal globin mRNA expression with neither significant cytotoxicity nor gene expression alteration of the examined globin regulators, KLF1, B-cell lymphoma/leukemia 11A and lymphoma/leukemia-related factor. These findings demonstrate that the downregulation of KLF4 is associated with increased embryonic and fetal globin gene expression in human erythroid progenitor cells. Moreover, identifying putative compounds or molecular approaches that effectively downregulate KLF4 and further induce embryonic globin expression may provide an alternative therapeutic strategy for α-globin substitution in severe α-thalassemia.

Fetal Hemoglobin Induction with Forced Chromatin Looping in Gene-Modified Erythroid Cells Differentiated from Rhesus Hematopoietic Progenitor Cells

Substantial advances in genome engineering offer new therapies for hemoglobin (Hb) disorders including sickle cell disease. Fetal hemoglobin reactivation remains a viable strategy, as recently demonstrated in human erythroid cells with an artificial looping molecule ZF-Ldb1, tethering Ldb1 to the γ-globin promoter by specific zinc finger proteins (Deng et al., 2014). Large animal preclinical testing can improve the therapeutic potential of such approaches by optimizing safety and efficacy issues before launching clinical trials in humans. A robust in vitro erythroid evaluation system, however, does not exist to determine if fetal hemoglobin reactivation can be modeled in the rhesus macaque. Therefore, in the current work, we aimed to develop an efficient in vitro erythroid differentiation model from rhesus hematopoietic progenitor cells with Hb production at detectable protein levels, and to evaluate rhesus fetal hemoglobin reactivation with ZF-Ldb1.To adapt a human erythroid differentiation method to rhesus cells, we used C3H or OP9 feeder cells to culture rhesus peripheral blood mononuclear cells (PBMCs) with HEMA differentiation media containing 10ng/ml stem cell factor (SCF), 1ng/ml interleukin-3 (IL3), 2U/ml erythropoietin (Epo), and 20% fetal bovine serum (FBS) (Migliaccio et al., 2010). Two weeks after erythroid differentiation, robust Hb production evaluated by HB electrophoresis was obtained on both C3H and OP9 feeder cells but not in feeder-free conditions. Increased concentration of SCF (20x), IL3 (10x), and Epo (1.5x) allowed for Hb production even in feeder-free conditions; however, fetal Hb was predominant (due to 20x SCF). Both conditions provided similar levels of enucleation (39±6.3% vs 29±5.6%, ns), indicating efficient differentiation. We then compared HEMA media with serum-free StemSpan-based differentiation media to evaluate transduction with a GFP-encoding lentiviral vector at MOI 2.5 two days after initiating differentiation. Transduction was almost completely inhibited (0.4±0.5%) in HEMA media, while StemSpan-based transduction media followed by HEMA differentiation media resulted in significant increase of transduction efficiency (41±8.5%, p<0.01). These data demonstrate that feeder cells are required for rhesus erythroid differentiation in HEMA media, while FBS strongly interferes lentiviral transduction.Next, we sought to establish a reverse phase HPLC method to quantitatively analyze rhesus globins among differentiated erythroid cells. Peaks on the HPLC chromatogram were eluted and run in liquid chromatography-mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) for protein identification. We identified β-globin and γ-globin (HBG1 and HBG2) peaks using both rhesus peripheral blood (99±26% and 0.65±0.28%) and in vitro differentiated rhesus erythroid cells (63±13% and 37±16%), which is essential for Hb switching analysis. In addition, human β-globin and T87Q β-globin (with anti-sickling mutation) proteins were also separated from rhesus globins in the HPLC chromatogram and were detectable for quantitation.Finally, we evaluated whether fetal Hb expression can be induced by the ZF-Ldb1 molecule in rhesus erythroid cells. We transduced differentiating rhesus CD34+ cells with ZF-Ldb1-encoding lentiviral vectors that are driven by an erythroid specific ankyrin-1 promoter, and contain GFP along with a β-globin locus control region (LCR) enhancer or YFP without the LCR. After erythroid differentiation, we obtained both LCR/ZF-Ldb1/GFP+ (9.4%) and ZF-Ldb1/YFP+ (1.3%) rhesus erythroid cells, and higher γ-globin RNA expression was detected for GFP+ and YFP+ fractions (68.1±1.5% and 59.8±2.1%, respectively), as compared to GFP- and YFP- controls (28.1±3.1% and 31.8±1.9%, p<0.05). These data demonstrate successful γ-globin induction with ZF-Ldb1 vectors in the rhesus macaque.In conclusion, we developed an efficient rhesus erythroid cell differentiation protocol from hematopoietic progenitor cells with robust Hb production detectable at protein levels, analyzed in a newly established HPLC method. The ZF-Ldb1 vector transduction allows for induction of γ-globin expression in rhesus erythroid cells. Further studies are warranted to optimize gene modification and transplantation of rhesus hematopoietic progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

A Genetic Variant Ameliorates β-Thalassemia Severity by Epigenetic-Mediated Elevation of Human Fetal Hemoglobin Expression

A delayed fetal-to-adult hemoglobin (Hb) switch ameliorates the severity of β-thalassemia and sickle cell disease. The molecular mechanism underlying the epigenetic dysregulation of the switch is unclear. To explore the potential cis-variants responsible for the Hb switching, we systematically analyzed an 80-kb region spanning the β-globin cluster using capture-based next-generation sequencing of 1142 Chinese β-thalassemia persons and identified 31 fetal hemoglobin (HbF)-associated haplotypes of the selected 28 tag regulatory single-nucleotide polymorphisms (rSNPs) in seven linkage disequilibrium (LD) blocks. A Ly1 antibody reactive (LYAR)-binding motif disruptive rSNP rs368698783 (G/A) from LD block 5 in the proximal promoter of hemoglobin subunit gamma 1 (HBG1) was found to be a significant predictor for β-thalassemia clinical severity by epigenetic-mediated variant-dependent HbF elevation. We found this rSNP accounted for 41.6% of β-hemoglobinopathy individuals as an ameliorating factor in a total of 2,738 individuals from southern China and Thailand. We uncovered that the minor allele of the rSNP triggers the attenuation of LYAR and two repressive epigenetic regulators DNA methyltransferase 3 alpha (DNMT3A) and protein arginine methyltransferase 5 (PRMT5) from the HBG promoters, mediating allele-biased γ-globin elevation by facilitating demethylation of HBG core promoter CpG sites in erythroid progenitor cells from β-thalassemia persons. The present study demonstrates that this common rSNP in the proximal Aγ-promoter is a major genetic modifier capable of ameliorating the severity of thalassemia major through the epigenetic-mediated regulation of the delayed fetal-to-adult Hb switch and provides potential targets for the treatment of β-hemoglobinopathy.

Induction of Fetal Hemoglobin Synthesis By Crispr/Cas9-Mediated Disruption of the β-Globin Locus Architecture

Naturally occurring, large deletions in the β-globin locus result in increased fetal hemoglobin (HbF) expression (HPFH, Hereditary Persistence of Fetal Hemoglobin), a condition that mitigates the clinical severity of Sickle Cell Disease (SCD) and β-thalassemia. Here, we integrated BCL11A and GATA1 transcription factor binding site analysis and HPFH mutational data to identify potential HbF silencers in the β-globin locus. Based on this analysis, we designed a CRISPR/Cas9 strategy to disrupt: (i) a 3.5-kb δγ-intergenic region that is specifically deleted in individuals with HPFH. This region contains BCL11A and GATA1 binding sites in adult, HbF-negative primary erythroblasts, thus representing a potential HbF silencer; (ii) a 7.2-kb region, encompassing the 3.5-kb δγ-intergenic element, which is the minimal naturally occurring deletion associated with HPFH ("Corfù" deletion; Chakalova et al., Blood, 2005); (iii) a 13.6-kb genomic region commonly deleted in HPFH, which includes the δ- and β-globin genes and putative intergenic HbF silencers. Targeted deletion or inversion of the 13.6-kb genomic region caused a substantial increase in γ-globin mRNA levels, re-activation of HbF synthesis and a concomitant decrease in HbA expression in adult human erythroid cells (HUDEP-2). Interestingly, deletion of the Corfù region or of the 3.5-kb putative HbF silencer, increased γ-globin gene transcription but failed to produce accumulation of γ-globin mRNA, suggesting a post-transcriptional regulation of γ-globin synthesis in the presence of an intact and active β-globin gene. We then tested re-activation of HbF synthesis in primary adult hematopoietic stem/progenitor cells differentiated towards the erythroid lineage in liquid and clonogenic cultures. Targeting the 13.6-kb genomic region resulted in a high proportion of γ-globin expressing primary erythroblasts, with HbF representing up to 50% of total hemoglobin. Cell morphology, erythroid marker profile, total hemoglobin levels and erythroid maturation were unaffected, consistent with the asymptomatic phenotype of adult HPFH carriers. These data suggest that this region could serve as target for therapeutic genome editing for HbF induction in β-hemoglobinopathies. Overall, this study contributes to the knowledge of the mechanisms underlying fetal to adult Hb switching, and provides clues for a genome editing approach to the treatment of SCD and β-thalassemia. DisclosuresCradick:CRISPR Therapeutics: Employment. Lundberg:CRISPR Therapeutics: Employment, Equity Ownership. Porteus:CRISPR Therapeutics: Consultancy, Equity Ownership. Mavilio:Adverum Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding.

559. Induction of Fetal Hemoglobin in Adult Erythroblasts by Genome Editing of the Beta-Globin Locus

Sickle cell disease (SCD) and β-thalassemia are severe anemias characterized by abnormal or reduced production of hemoglobin β-chains. Autologous transplantation of genetically corrected hematopoietic stem cells (HSC) is an attractive therapeutic alternative for patients lacking a compatible HSC donor. Naturally occurring, large deletions in the β-globin locus result in increased fetal hemoglobin (HbF) expression (HPFH, Hereditary Persistence of Fetal Hemoglobin), a condition that mitigates the clinical severity of β-hemoglobinopathies. Here, we integrated BCL11A and GATA1 transcription factor binding site analysis and HPFH mutational data to identify potential HbF silencers in the β-globin locus. Based on this analysis, we designed a CRISPR/Cas9 strategy to disrupt a 13-kb genomic region commonly deleted in HPFH, which includes the Δ- and β-globin genes and putative intergenic HbF silencers, and achieved efficient targeted deletion in erythroid cell lines by plasmid, RNA and lentiviral delivery of the CRISPR/Cas9 nuclease system. RT-PCR showed a dramatic increase in γ-globin mRNA levels in modified adult hematopoietic stem progenitor cells (HSPC)-derived erythroid cell lines (HUDEP-2). FACS and HPLC analysis demonstrated reactivation of HbF and a concomitant decrease in HbA expression. Cell morphology, erythroid marker profile, total Hb levels and erythroid maturation were unaffected, consistent with the asymptomatic phenotype of adult HPFH carriers. The same strategy was tested in primary human erythroblasts by lentiviral transduction of adult CD34+ HSPCs followed by in vitro erythroid differentiation in liquid and clonogenic cultures. Deletion of potential HbF silencers resulted in a 35% increase in γ-globin expression compared to basal levels in primary human erythroblasts as measured by HPLC, suggesting that these sequences could serve as targets for therapeutic genome editing for HbF induction in β-hemoglobinopathies. We are currently testing the efficiency of our CRISPR/Cas9-based strategy in patient-derived HSPCs. Overall, this study contributes to the knowledge of the mechanisms underlying fetal to adult Hb switching, and provides clues for a therapeutic strategy for SCD and β-thalassemia.

Gene Expression Analysis of the Brazilian Type of Hereditary Persistence of Fetal Hemoglobin: Identification of Genes that Could be Related to γ-Globin Activation

Increased γ-globin production and consequent fetal hemoglobin (Hb F, α2γ2) formation is an important modulator of the clinical and hematological features of hemolytic anemias, such as sickle cell disease and β-thalassemia (β-thal). Hb F genes are genetically regulated, but despite numerous studies, the molecular basis of hemoglobin (Hb) switching is not completely understood. Hereditary persistence of fetal Hb (HPFH) is a consequence of impaired switching in adult life, which results in the continued expression of the γ-globin gene. This study was undertaken to identify genes that could be involved in Hb switching and/or maintenance of elevated Hb F levels. Two libraries were constructed using reticulocytes from normal donors and from Brazilian HPFH subjects. Results suggest that the maintenance of Hb F levels could be associated with some gene/protein expression modifications, such as low expression of KLF1, a transcription factor known to contribute to the regulation and modulation of Hb switching, decreased expression of MIER1, known for the recruitment of chromatin remodeling enzymes, and decreased expression of HOOK3. These data suggest new genes that may play a role in globin gene regulation, γ-globin gene expression and augmentation of Hb F levels, and may represent newly-defined cellular pathways for the control of Hb switching in erythroid cells.

Mechanism of human Hb switching: a possible role of the kit receptor/miR 221-222 complex

The human hemoglobin switch (HbF-->HbA) takes place in the peri/post-natal period. In adult life, however, the residual HbF (<1%) may be partially reactivated by chemical inducers and/or cytokines such as the kit ligand (KL). MicroRNAs (miRs) play a pivotal role in normal hematopoiesis: downmodulation of miR-221/222 stimulates human erythropoietic proliferation through upmodulation of the kit receptor. We have explored the possible role of kit/KL in perinatal Hb switching by evaluating: i) the expression levels of both kit and kit ligand on CD34(+) cells and in plasma isolated from pre-, mid- and full-term cord blood samples; ii) the reactivation of HbF synthesis in KL-treated unilineage erythroid cell cultures; iii) the functional role of miR-221/222 in HbF production. In perinatal life, kit expression showed a gradual decline directly correlated to the decrease of HbF (from 80-90% to <30%). Moreover, in full-term cord blood erythroid cultures, kit ligand induced a marked increase of HbF (up to 80%) specifically abrogated by addition of the kit inhibitor imatinib, thus reversing the Hb switch. MiR-221/222 expression exhibited rising levels during peri/post-natal development. In functional studies, overexpression of these miRs in cord blood progenitors caused a remarkable decrease in kit expression, erythroblast proliferation and HbF content, whereas their suppression induced opposite effects. Our studies indicate that human perinatal Hb switching is under control of the kit receptor/miR 221-222 complex. We do not exclude, however, that other mechanisms (i.e. glucocorticoids and the HbF inhibitor BCL11A) may also contribute to the peri/post-natal Hb switch.

Role Of Stem Cell Factor In The Reactivation Of Human Fetal Hemoglobin

In humans the switch from fetal to adult hemoglobin (HbF → HbA) takes place in the perinatal and postnatal period, determining the progressive replacement of HbF with HbA synthesis (i.e., the relative HbF content in red blood cells decreases from 80–90% to <1%). In spite of more than twenty years of intensive investigations on this classic model, the molecular mechanisms regulating the Hb switching, as well as HbF synthesis in adults, has been only in part elucidated. In adult life, the residual HbF, restricted to F cell compartment, may be reactivated up to 10–20% of total Hb synthesis in various conditions associated with “stress erythropoiesis”: this reactivation represented until now an interesting model of partial Hb switch reverse with important therapeutic implications in patients with hemoglobinopathies, and particularly in β-thalassemia. In vitro and in vivo models have led to the identification of several chemical compounds able to reactivate HbF synthesis in adult erythroid cells. Although the impact of these HbF inducers, including hypomethylating agents, histone deacetylase inhibitors and hydroxyurea, was clear on the natural history of sickle cell anemia, the benefit on the clinical course of β-thalassemia was only limited: particularly, the toxicity and the modest increase in γ-globin reactivation indicated the need for improved agents able to induce higher levels of HbF. In the present review we describe the biologic properties of Stem Cell Factor (SCF), a cytokine sustaining the survival and proliferation of erythroid cells, that at pharmacological doses acts as a potent stimulator of HbF synthesis in adult erythroid cells.

Epigenetic Analysis of Beta-Globin Gene Cluster during Hematopoiesis.

Hemoglobin (Hb) switching is described as temporal, tissue- and stage-specific patterns of globin gene expression; from embryonic to fetal and adult Hb in parallel to developmental stages of erythropoiesis. DNA methylation, one of the epigenetic mechanisms, was associated with inactivated chromatin domain and repressive transcription. To study the role of the DNA methylation on the beta (β)-globin genes, we analyzed CpG dinucleotides in 87 kb regions around β-globin gene cluster, including 5'upstream locus control regions (LCR; DNAse I Hypersensitive site (HS) 1–5), 3'HS1, the promoter regions of the G-and A-gamma (Gγ and Aγ), and β-globin genes, in several representative cells. These cells wereprimary adult erythroid cells culture (three different stages: early, intermediate, and late),fetal cord blood DNA, andneutrophil cell line (non-erythroid).Using bisulphite modification, followed by nested PCR and in vitro translation, the cleavage products were analysed by MALDI-TOF Mass Spectrometry to quantify the DNA methylation level. The results were consistent with bisulphite sequencing. We found that the promoters of Gγ and Aγ-globin genes were significantly hypomethylated in fetal cells (44% and 47% global methylation), when γ-globin genes were fully expressed, while they were heavily methylated in non-erythroid (86% and 95%). There was also a decreasing trend of the DNA methylation level at Gγ and Aγ-globin genes during adult erythroid differentiation from 80% and 82%, in early stage, to 67% and 66% in late stage (p=0.12 and 0.04). At β-globin promoter, the global methylation level changed from 90% in non-erythroid to 81%, 42%, and 26% in fetal, early and late adult erythroid cells, respectively. Moreover, we found the significant changes at 5'HS4, 3, and 1 as all erythroid cells were hypomethylated compare to non-erythroid. While at the insulators, 5'HS5 and 3'HS1, all tested CpG dinucleotides were heavily methylated in all cells. This is the first report that demonstrates the differences in DNA methylation at β-globin LCR between erythroid and non-erythroid cells. These epigenetic marks were associated with globin genes expression and might be useful to predict clinical severity in patients with β-thalassemia intermedia.

Disorders of the synthesis of human fetal hemoglobin

Fetal hemoglobin (HbF), the predominant hemoglobin in the fetus, is a mixture of two molecular species (alpha(2)(G)gamma(2) and alpha(2)(A)gamma(2)) that differ only at position 136 reflecting the products of two nonallelic gamma-globin genes. At the time of birth, HbF accounts for approximately 70% of the total Hb. The (G)gamma:(A)gamma globin ratio in the HbF of normal newborn is 70:30 whereas in the trace amounts of HbF that is found in the adult it reverses to 40:60 because of a gamma- to beta-globin gene switch. Alterations of these ratios are indicative of a molecular defect at the level of the HbF synthesis. Qualitative hemoglobinopathies due to (G)gamma and (A)gamma chain structural variants, and quantitative hemoglobinopathies affecting the synthesis of HbF such as gamma-thalassemias, duplications, triplications, and even sextuplications of the gamma-globin genes, which may be detected in newborn blood lysates, have been described. Moreover, several pathological and nonpathological conditions affecting the beta-globin gene cluster, such as beta-thalassemia, sickle cell disease, deltabeta-thalassemia, and hereditary persistence of HbF syndromes, are characterized by the continued synthesis of gamma-globin chains in the adult life. Studies of these natural mutants associated with increased synthesis of HbF in adult life have provided considerable insight into the understanding of the control of globin gene expression and Hb switching.

Ferritin Heavy Chain Stimulates HbS-to-HbF Switching in Erythroid Precursor Cells from Sickle Cell Patients.

We have found that ferritin heavy chain (FtH), an antioxidant/stress response/iron-storage protein, localizes to the nucleus in K562 cells and represses the human adult beta-globin promoter in transient assays in primate cells (Broyles et al., PNAS 98: 9145, 2001). Since other work indicates FtH is also a gene activator of fetal-globin genes, we hypothesize that FtH is a long-sought developmental hemoglobin (Hb) switching factor and that delivery of FtH to human adult erythroid cell precursors will reverse the phenotype to HbF, offering a phenotypic cure for sickle cell disease (SCD). Chromatin immunoprecipitation (ChIP) assays, antisense treatments, and an FtH transgenic mouse have confirmed that FtH is a globin gene regulatory protein in vivo. With erythroid precursor cells from pediatric SCD patients, under an IRB-approved protocol, we have used a two-phase culture system for in vitro maturation of erythroid cells in the presence of FtH, delivered to the cells as pure protein, as an expression plasmid, or as a priority inducer compound that activates the endogenous FtH gene. HPLC with a PolyCAT A column was used to separate and quantify human Hbs. With each mode of delivery, FtH stimulated a complete switch from HbS to HbF. This result was repeatable in multiple experiments using erythroid precursor cells from three different SCD donors. Fluorescently-labeled recombinant human FtH protein was taken into red cell precursors in culture, suggesting that the purified protein can be directly delivered without gene therapy. This method of producing a phenotypic cure in SCD patients should be easy and inexpensive to deliver in vivo.

The effect of hypoxia and stem cell source on haemoglobin switching

This study investigated whether relative changes that accompany the naturally occurring shifts in haematopoietic sites during human development play a role in haemoglobin (Hb) switching or whether Hb switching is innately programmed into cells. CD34(+)/Lineage(-) haematopoietic stem/progenitor cells (HSCs) were isolated from human fetal liver (F-LVR), cord blood (CB), and adult bone marrow (ABM), and the Hb was characterized by flow cytometry on cultures that generated enucleated red cells. All feeder layers (stroma from F-LVR, ABM, and human fetal aorta) enhanced cell proliferation and erythropoiesis but did not affect Hb type. HSCs from CB and F-LVR generated the same Hb profile under normoxia and hypoxia. HSCs from ABM had single-positive HbA and double-positive HbA and HbF cells at normoxia and almost entirely double-positive cells at hypoxia. Further characterization of these ABM cultures was determined by following mRNA expression for the transcription factors erythroid Kruppel-like factor (EKLF) and fetal Kruppel-like factor (FKLF) as a function of time in cultures under hypoxia and normoxia. The erythroid-specific isoform of 5-amino-levulinate synthase (ALAS2) was also expressed under hypoxic conditions. We conclude that Hb switching is affected by the environment but not all HSCs are preprogrammed to respond.

Neonatal hyperbilirubinemia in Japanese neonates: analysis of the heme oxygenase-1 gene and fetal hemoglobin composition in cord blood.

Neonatal hyperbilirubinemia is frequent and severe in Japanese infants. Although the G71R mutation of the bilirubin uridine diphosphate-glucuronosyltransferase gene is associated with severe neonatal hyperbilirubinemia in this population, it accounts for only half of the neonates with severe hyperbilirubinemia. It was suggested that increased bilirubin production would also be associated with severe neonatal hyperbilirubinemia in Japanese infants. To elucidate the genetic factors causing severe hyperbilirubinemia in these patients, we studied two notable factors associated with bilirubin production: heme oxygenase-1, a key enzyme of heme metabolism, and fetal Hb composition, a factor possibly associated with heme load in neonates. We first determined the sequences of promoter and all coding regions of the heme oxygenase-1 gene in Japanese neonates who had undergone phototherapy, but found no mutation except for the polymorphic (GT)n repeats in the promoter region. These repeats modulate the transcription of the heme oxygenase-1 gene, and the longer repeat sequences are known to reduce the transcription. We detected a significant difference in the allele frequencies of each number of (GT)n repeats between Japanese and German populations. However, we could not find a relation between those polymorphisms and neonatal hyperbilirubinemia. We next analyzed the state of Hb switching of the gamma- to beta-globin chain and the phenotype of gamma-globin chain isoforms in cord blood. We found no relation between fetal Hb composition and neonatal hyperbilirubinemia. Further studies are required to elucidate genetic or environmental factors in neonatal hyperbilirubinemia in Japanese infants.

Erythropoiesis and Conversion of RBCs and Hemoglobins from Larval to Adult Type during Amphibian Development

Abstract In anuran amphibians transitions of hemoglobins (Hbs) and red blood cells (RBCs) from the larval to the adult type have been reported to occur at metamorphosis, depending on certain influence of thyroid hormones (THs). Contrary to this, transition of RBCs/Hbs from the larval to the adult type during the metamorphosis in a urodele, Hynobius retardatus occurs almost independently of thyroid activity, but dependent on certain pituitary factor(s). All findings reported so far support the idea that the Hb switching in H. retardatus occurs in a single RBC population (“Hb switching” model), rather than the concept that larval RBCs are replaced by new, adult RBCs (“RBC replacement” model) as is known to occur in many anurans. Erythropoiesis in vertebrates occurs with two distinct phases, termed primitive and definitive. Primitive erythropoiesis generally provides embryonic/larval erythroids, and definitive hematopoiesis contributes to adult RBCs. Primitive erythropoiesis in Xenopus laevis occurs in the v...

Mismatch repair and virulence

Neisseria meningitidis can produce such an extensive variation of adhesive, invasive and antigenic properties that a single infecting organism can give rise to diverse populations with very different virulence properties. Such a strategy increases the probability of generating a variant that is better adapted to the new host. The variability of N. meningitidis virulence determinants is generated very efficiently by frame-shift mutagenesis in chromosomal regions containing repeated nucleotide tracts called contingency loci. The frequency of such mutagenesis among N. meningitidis clinical isolates is highly variable. For example, the frequency of phase variation in hemoglobin (Hb) receptors among clinical isolates of serogroups A, B and C ranges from 10−6 to 10−2.Now, Richardson and Stojiljkovic 1xMismatch repair and the regulation of phase variation in Neisseria meningitidis. Richardson, A.R. and Stojiljkovic, I. Mol. Microbiol. 2001; 40: 645–655Crossref | PubMed | Scopus (95)See all References1 have suggested that the frequency of phase variation might be genetically determined for a particular strain, as they found two unlinked Hb receptors underwent phase variation with similar frequencies within a given isolate. They also observed that an increase in the frequency of phase variation was often accompanied by high rates of base-substitution mutations conferring resistance to rifampicin and nalidixic acid. This formidable genetic instability was shown to be a consequence of defects in the mismatch-repair genes. Inactivation of mismatch repair increases the Hb switching frequency 250–500-fold and base substitution mutagenesis 20–30-fold. These results, together with the previous observation that Pseudomonas aeruginosa strains isolated from lungs of cystic fibrosis patients have a very high frequency of mismatch-repair-deficient mutants, which were much more resistant to antibiotics than non-mutated pathogenic isolates 2xHigh frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Oliver, A. et al. Science. 2000; 288: 1251–1254Crossref | PubMed | Scopus (811)See all References2, suggest that high mutation rates could increase the virulence of pathogenic strains.

Mismatch repair and the regulation of phase variation in Neisseria meningitidis.

Neisseria meningitidis controls the expression of several genes involved in host adaptation by a process known as phase variation. The phase variation frequency of haemoglobin (Hb) receptors among clinical isolates of serogroups A, B and C differed drastically, ranging from approximately 10(-6) to 10(-2) cfu-1. Frequencies of phase variation are a genetic trait of a particular strain, as two unlinked Hb receptors, hpuAB and hmbR, phase varied with similar frequencies within a given isolate. Based on these frequencies, six Neisserial clinical isolates could be grouped into three distinct classes; slow, medium and fast. An increase in phase variation frequency was accompanied by high rates of spontaneous mutation to rifampicin and nalidixic acid resistance in one medium and one fast strain. The remaining three medium strains displayed elevated levels of phase variation without increases in overall mutability, as they possessed low rates of spontaneous mutation to drug resistance. The mismatch repair system of N. meningitidis was found to play an important role in determining the overall mutability of the clinical isolates. Inactivation of mismatch repair in any strain, regardless of its original phenotype, increased mutability to a level seen in the fast strain. Insertional inactivation of mutS and mutL in the slow strain led to 500- and 250-fold increases in hmbR switching frequency respectively. Concurrently, the frequency of spontaneous point mutations of mutS and mutL mutants from the slow strain was increased 20- to 30-fold to the level seen in the high strain. The status of Dam methylation did not correlate with either the phase variation frequency of Hb receptors or the general mutability of Neisserial strains. Analysis of an expanded set of isolates identified defects in mismatch repair as the genetic basis for strains displaying both the fast Hb switching and high mutation rate phenotypes. In conclusion, elevated frequencies of phase variation were accompanied by increased overall mutability in some N. meningitidis isolates including strains shown to be mismatch repair defective. Other isolates have evolved mechanisms that seem to affect only the switching frequency of phase-variable genes without an accompanied increased accumulation of spontaneous mutations.

Contribution of Ventral and Dorsal Mesoderm to Primitive and Definitive Erythropoiesis in the Salamander Hynobius retardatus

Previously, we found that the conversion of hemoglobins (Hbs) from the larval to the adult type occurred within a single erythroid cell population in a salamander, Hynobius retardatus (“Hb switching” model), whereas the transition involves replacement of red-blood-cell (RBC) populations (“RBC replacement” model) in many amphibians (M. Yamaguchi, H. Takahashi, and M. Wakahara, 2000, Dev. Gene Evol. 210, 180–189). To further characterize the Hb transition, developmental changes in the erythropoietic sites have been intensively analyzed using larval- and adult-specific globin antibodies and globin and GATA-3 RNA probes. Cells of the ventral blood island (VBI) and the dorsolateral plate (DLP) in embryos differentiate in situ to erythroid cells that contain larval globin mRNA, suggesting that both the VBI and the DLP contribute to “primitive” erythropoiesis. In contrast, the expression pattern of the GATA-3 gene suggests that cells of the DLP may contribute to “definitive” hematopoiesis. In order to determine whether it is possible to define a definitive erythropoiesis in H. retardatus or not, further experiments were done: (1) when metamorphosing larvae were treated with phenylhydrazine to induce anemia and then bled at the postmetamorphic stage after recovery from the anemia, a precocious Hb transition was observed in these animals; (2) an RBC population expressing only adult Hb was confirmed by subtracting the number of RBCs expressing larval Hb from the total number of RBCs during metamorphosis. All these results support the existence of a definitive erythroid cell population that contributes only adult RBCs in this species.