The First Trimester Human Placenta Is An Embryonic Hematopoietic Organ with An Unexpected Function in Primitive Erythroid Maturation.

Abstract Abstract 1508 Poster Board I-531 Developmental hematopoiesis satisfies the immediate needs of the embryo and provides the hematopoietic stem cells necessary for lifelong blood homeostasis. Recently, the mid-gestation mouse placenta was identified as an important definitive hematopoietic organ; data from us and others indicates that the human placenta functions analogously, harboring and potentially generating hematopoietic stem and progenitor cells. The function of the human placenta in embryonic hematopoiesis has not been addressed. We assessed the hematopoietic potential of placental tissues before the onset of feto-placental circulation and found robust de novo generation of clonogenic progenitors. Interestingly, pre-circulation progenitors were greatly enriched (69 ± 14% of total) in macrophage progenitors. Immunostaining demonstrated that these progenitors are generated in the chorionic plate. As development progresses, placental macrophages migrate to the villous stroma. Surprisingly, in the villi, placental macrophages associate closely with an unexpected population of extravascular, z-globin+ primitive erythroid cells, prompting the hypothesis that embryonic macrophages promote the maturation of primitive erythroblasts in the placenta. Concordantly, we found that human primitive erythroblasts enucleate, as has been recently demonstrated in mice. Interestingly, the first enucleated erythroid cells were found in the villous stroma; between 5-7 weeks developmental age, their relative frequency in the stroma was 2-4 fold higher than in lumens. We also observed free nuclei in the placental stroma; a similar population of ejected red cell nuclei, termed pyrenocytes, has recently been described in mouse embryos. Immunohistochemistry and FACS confirmed that these free nuclei in the placenta were pyrenocytes. Electron microscopy revealed placental macrophages containing ingested red cell nuclei. Together, this data suggests that placenta-derived macrophages promote the terminal maturation of primitive erythroblasts in the villous stroma, nominating the first trimester human placenta as a site of primitive hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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The first trimester human placenta is a site for terminal maturation of primitive erythroid cells

Blood ◽

10.1182/blood-2010-04-279489 ◽

2010 ◽

Vol 116 (17) ◽

pp. 3321-3330 ◽

Cited By ~ 56

Author(s):

Ben Van Handel ◽

Sacha L. Prashad ◽

Nargess Hassanzadeh-Kiabi ◽

Andy Huang ◽

Mattias Magnusson ◽

...

Keyword(s):

Human Placenta ◽

Blood Cells ◽

Fetal Liver ◽

First Trimester ◽

Erythroid Cells ◽

Chorionic Plate ◽

Placental Villi ◽

Placental Macrophages ◽

A Site ◽

Broad Role

Abstract Embryonic hematopoiesis starts via the generation of primitive red blood cells (RBCs) that satisfy the embryo's immediate oxygen needs. Although primitive RBCs were thought to retain their nuclei, recent studies have shown that primitive RBCs in mice enucleate in the fetal liver. It has been unknown whether human primitive RBCs enucleate, and what hematopoietic site might support this process. Our data indicate that the terminal maturation and enucleation of human primitive RBCs occurs in first trimester placental villi. Extravascular ζ-globin+ primitive erythroid cells were found in placental villi between 5-7 weeks of development, at which time the frequency of enucleated RBCs was higher in the villous stroma than in circulation. RBC enucleation was further evidenced by the presence of primitive reticulocytes and pyrenocytes (ejected RBC nuclei) in the placenta. Extravascular RBCs were found to associate with placental macrophages, which contained ingested nuclei. Clonogenic macrophage progenitors of fetal origin were present in the chorionic plate of the placenta before the onset of fetoplacental circulation, after which macrophages had migrated to the villi. These findings indicate that placental macrophages may assist the enucleation process of primitive RBCs in placental villi, implying an unexpectedly broad role for the placenta in embryonic hematopoiesis.

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Placenta Is a Niche for Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v104.11.2671.2671 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2671-2671

Author(s):

Hanna K.A. Mikkola ◽

Christos Gekas ◽

Francoise Dieterlen-Lievre ◽

Stuart H. Orkin

Keyword(s):

De Novo ◽

Fetal Liver ◽

Yolk Sac ◽

Mouse Development ◽

Adult Type ◽

Cell Potential ◽

Hematopoietic Stem ◽

Hematopoietic Organ ◽

Hematopoietic System ◽

Hematopoietic Organs

Abstract The hematopoietic system in the embryo develops in anatomically distinct sites, facilitating rapid generation of erythroid cells and formation of a pool of pluripotent HSCs. The origin of definitive HSCs is not fully resolved, and little is known about how the different fetal hematopoietic microenvironments direct the genesis, maturation, expansion and differentiation of HSCs. In avians, de novo hematopoiesis occurs not only in the yolk sac and the AGM but also in another mesodermal appendage, the allantois. In mammals, the allantois forms the umbilical cord and fetal placenta upon fusion with the chorion. The placenta has not been recognized as a hematopoietic organ, although Melchers reported fetal B-cell potential in murine placenta 25 years ago (Nature 1979, 277:219). Recently, Alvarez-Silva et al. showed that the placenta is a rich source for multipotential hematopoietic progenitors prior to the fetal liver (Development2003, 130:5437). We have performed spatial and temporal analysis of HSCs during mouse development and for the first time assessed HSC activity in the placenta. Hematopoietic organs from E10.5-18.5 embryos (CD45.1/CD45.2) were treated with collagenase and transplanted in limiting dilutions (3–1/1000 embryo equivalents, ee) into irradiated CD45.2+ adult hosts with CD45.1+ support BM cells. Reconstitution was analyzed by FACS and HSCs were quantified as repopulating units (RUs/ee = ([reconstituted recipients] /[total recipients]) /[transplanted dose]). Our data show that the placenta functions as a hematopoietic organ that during midgestation harbors a large pool of pluripotent HSCs. The onset of HSC activity in the placenta parallels that of the AGM starting at E10.5–11.0. However, the placenta HSC pool expands until E12.5–13.5 (>50 RUs) contrasting lack of HSC expansion in the AGM. The expansion of CD34+c-kit+ HSCs in the placenta occurs prior to and during the initial expansion of HSCs in the fetal liver and is not accompanied with myeloerythroid differentiation. A far greater expansion of placenta HSCs compared to that of clonogenic progenitors (17-fold vs. 2-fold at E11.5–12.5) suggests that the placenta provides a favorable niche for HSCs. Indeed, placenta HSCs possess functional properties of authentic adult-type HSCs by providing high level multilineage reconstitution for >5 months and exhibiting self-renewal capacity upon serial transplantation. Importantly, placenta HSCs are distinct from circulating HSCs that appear in low numbers after E11.5. HSC activity in the placenta declines towards the end of gestation while HSCs in the fetal liver and blood continue to increase, possibly reflecting mobilization of placenta HSCs to the fetal liver and other developing hematopoietic organs. The early onset of HSC activity in the placenta suggests that the allantois and its derivatives may participate in de novo genesis and maturation of HSCs together with the AGM and possibly the yolk sac. As the main blood volume from the dorsal aorta reaches the fetal liver via umbilical vessels and the placenta, placenta may also provide a niche where nascent HSCs, or pre-HSCs, from the AGM colonize for maturation and expansion prior to seeding fetal liver. While further studies are needed to define the precise origin of placenta HSCs and the function of placenta microenvironment as an HSC supportive niche, the unique kinetics and magnitude of HSC activity suggest an important, previously unappreciated role for the placenta in establishing the definitive hematopoietic system.

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The First Trimester Human Placenta Is a Site of Primitive Red Blood Cell Maturation.

Blood ◽

10.1182/blood.v110.11.2224.2224 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2224-2224

Author(s):

Benjamin J. Van Handel ◽

Sacha Prashad ◽

Andy Huang ◽

Eija Hamalainen ◽

Angela Chen ◽

...

Keyword(s):

Bone Marrow ◽

Red Blood Cells ◽

Human Placenta ◽

Blood Cells ◽

Fetal Liver ◽

First Trimester ◽

Yolk Sac ◽

Primitive Hematopoiesis ◽

Placental Macrophages ◽

Definitive Erythropoiesis

Abstract Embryonic hematopoiesis occurs in multiple anatomic sites and is generally divided into two waves, primitive and definitive. The primitive wave produces mostly red blood cells in the yolk sac, while the definitive wave generates hematopoietic stem cells (HSCs) that provide lifelong blood homeostasis. Definitive erythropoiesis, occurring first in the fetal liver and eventually the bone marrow, is an orchestrated process in which erythroblasts cluster around a central macrophage. These functional units, termed erythroblast islands, facilitate the maturation of nucleated erythroblasts to enucleated erythrocytes. It has long been thought that primitive red cells maintain their nucleus until undergoing apoptosis; however, the enucleation of primitive erythroblasts has been recently documented in mice, although the site at which this occurs is unknown. We have recently identified the placenta as a major hematopoietic organ that promotes the development of HSCs in mice; preliminary data suggests that the first trimester human placenta also supports definitive hematopoiesis. Surprisingly, our most recent findings indicate a novel, unexpected role for the human placenta in primitive hematopoiesis: the promotion of terminal maturation of primitive erythroblasts. Analysis of placental sections revealed a striking tendency of primitive red blood cells to extravasate from blood vessels in the villi and migrate out into the stroma. Furthermore, once out in the stroma, primitive erythroblasts mature: they lose expression of CD43 and enucleate. The finding that human primitive red blood cells enucleate is undocumented; interestingly, the developmental timing of erythroblast enucleation in humans parallels that in mice. At three weeks, nascent vessels in the placenta are empty, but starting at about 4 weeks, placental circulation begins and fills these vessels with large, nucleated primitive erythroblasts generated in the yolk sac. The migration of primitive erythroblasts into the stroma occurs between 4.5 and 7 weeks. Enucleation mirrors this process, with a large enrichment of enucleated cells in the stroma versus in the vessels at early developmental ages, suggesting that primitive erythroblasts enucleate in the placental stroma. This phenomenon is restricted to placental villi and does not occur in the chorionic plate. Strikingly, extravasated erythroblasts are often in close proximity to placental macrophages, reminiscent of the macrophage-erythroblast associations seen in fetal liver and bone marrow erythropoiesis at later developmental stages. Fetal liver-derived definitive erythrocytes enter circulation at around 8 weeks. After 9–10 weeks, most red blood cells can be observed in vessels, and almost all are enucleated. The concerted processes of extravasation and maturation of primitive erythroblasts in placental stroma nominate the placenta as an important site in primitive hematopoiesis. Furthermore, the association between placental macrophages and primitive erythroblasts suggests that primitive and definitive erythropoiesis share common mechanisms of terminal maturation.

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Recreating a Human Hematopoietic Stem Cell Niche in Vitro by Unique Stroma Cells from the First Trimester Human Placenta and Fetal Liver

Blood ◽

10.1182/blood.v112.11.3568.3568 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3568-3568

Author(s):

Mattias Magnusson ◽

Melissa Romero ◽

Sacha Prashad ◽

Ben Van Handel ◽

Suvi Aivio ◽

...

Keyword(s):

Stem Cells ◽

Human Placenta ◽

Ex Vivo ◽

Fetal Liver ◽

Embryonic Stem ◽

Cell Types ◽

First Trimester ◽

Hematopoietic Stem ◽

Human Hscs

Abstract Expansion of human hematopoietic stem cells (HSCs) ex vivo has been difficult due to limited understanding of their growth requirements and the molecular complexity of their natural microenvironments. To mimic the niches in which human HSCs normally develop and expand during ontogeny, we have derived two unique types of stromal niche cells from the first trimester human placenta and the fetal liver. These lines either support maintenance of multipotential progenitors in culture, or promote differentiation into macrophages. Impressively, the supportive lines facilitate over 50,000-fold expansion of the most immature human HSCs/progenitors (CD34+CD38-Thy1+) during 8-week culture supplemented with minimal cytokines FLT3L, SCF and TPO, whereas the cells cultured on non-supportive stroma or without stroma under the same conditions differentiated within 2 weeks. As the supportive stroma lines also facilitate differentiation of human hematopoietic progenitors into myeloid, erythroid and B-lymphoid lineages, we were able to show that the expanded progenitors preserved full multipotentiality during long-term culture ex vivo. Furthermore, our findings indicate that the supportive stroma lines also direct differentiation of human embryonic stem cells (hESC) into hematopoietic progenitor cells (CD45+CD34+) that generate multiple types of myeloerythroid colonies. These data imply that the unique supportive niche cells can both support hematopoietic specification and sustain a multilineage hematopoietic hierarchy in culture over several weeks. Strikingly, the supportive effect from the unique stromal cells was dominant over the differentiation effect from the non-supportive lines. Even supernatant from the supportive lines was able to partially protect the progenitors that were cultured on the non-supportive lines, whereas mixing of the two types of stroma resulted in sustained preservation of the multipotential progenitors. These results indicate that the supportive stroma cells possess both secreted and surface bound molecules that protect multipotentiality of HSCs. Global gene expression analysis revealed that the supportive stroma lines from both the placenta and the fetal liver were almost identical (r=0.99) and very different from the non-supportive lines that promote differentiation (r=0.34), implying that they represent two distinct niche cell types. Interestingly, the non-supportive lines express known mesenchymal markers such as (CD73, CD44 and CD166), whereas the identity of the supportive cells is less obvious. In summary, we have identified unique human stromal niche cells that may be critical components of the HSC niches in the placenta and the fetal liver. Molecular characterization of these stroma lines may enable us to define key mechanisms that govern the multipotentiality of HSCs.

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L-Leucine Improves the Anemia of the 5q- Syndrome Via Activation of the mTOR (mammalian target of rapamycin) Pathway

Blood ◽

10.1182/blood.v118.21.970.970 ◽

2011 ◽

Vol 118 (21) ◽

pp. 970-970

Author(s):

Maria Virgilio ◽

Elspeth Payne ◽

Anupama Narla ◽

Hong Sun ◽

Barry H. Paw ◽

...

Keyword(s):

Conflicts Of Interest ◽

Mammalian Target Of Rapamycin ◽

Macrocytic Anemia ◽

Flow Cytometric Analysis ◽

Ribosomal Subunit ◽

Mrna Translation ◽

Target Of Rapamycin ◽

Translational Efficiency ◽

Erythroid Cells ◽

Hematopoietic Stem

Abstract Abstract 970 The “5q- syndrome” is a subtype of myelodysplastic syndrome (MDS) characterized by a profound macrocytic anemia that is thought to arise from the heterozygous loss of the RPS14 gene on chromosome 5q. The 5q- syndrome shares many characteristics with another ribosomopathy, Diamond Blackfan anemia (DBA), wherein half the patients have a heterozygous loss of a ribosomal protein gene, with RPS19 being the most frequently mutated. Both RPS19 and RPS14 are components of the 40S small ribosomal subunit. Heterozygous loss of both genes is thought to contribute to the pathophysiology of these ribosomopathies by impairing ribosome function and thereby decreasing mRNA translational efficiency. L-Leucine, a branched-chain amino acid has been shown to be a potent stimulator of mRNA translation. In a case report, administration of L-Leucine to a DBA patient resulted in complete recovery of the anemia and improvement in appetite and growth parameters. We therefore hypothesized that L-Leucine could improve the anemia associated with heterozygous loss of RPS14. To test this hypotheisis, we used zebrafish as an in vivo model for del(5q) MDS. Using antisense morpholinos, we knocked down Rps14 expression to haploinsufficient levels and observed anemia in the resulting morphants. Treatment of these morphants with L-Leucine, but not D-Leuicne, resulted in a dramatic increase in hemoglobinization as well as an increase in total erythroid cells. This observation was further validated in vitro using human CD34+ hematopoietic progenitor cells infected with RPS14 shRNA. Flow cytometric analysis demonstrated that treatment with L-Leucine increased the total number of erythroid cells (glycophorin-A/CD71 expressing cells) compared to untreated cells. In previous studies, L-leucine has been shown to upregulate mRNA translation by activating the mTOR (mammalian target of rapamycin) pathway and its downstream targets S6Kinase (S6K1) and 4E-Binding proteins (4E-BPs). We demonstrated increased levels of phospho-S6K1 as a result of L-Leucine treatment in the rps14 zebrafish morphant embryos. This increased phosphorylation of S6K1 was inhibited by rapamycin, suggesting specificity of mTOR activation in bringing about the L-Leucine effect. In summary, we have successfully demonstrated, in both a zebrafish model and in human primary hematopoietic stem cells, that L-Leucine alleviates the anemia associated with del(5q) MDS. This effect is likely mediated by activation of the mTOR pathway resulting in increased mRNA translation and an improvement in the anemia associated with loss of RPS14. Our studies support the further evaluation of L-Leucine as a potential therapeutic agent in the treatment of the 5q- syndrome. Disclosures: No relevant conflicts of interest to declare.

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From Hematopoietic Stem Cell to Erythroblast: Regulation of Red Cell Production At Multiple Levels

Blood ◽

10.1182/blood.v118.21.sci-1.sci-1 ◽

2011 ◽

Vol 118 (21) ◽

pp. SCI-1-SCI-1 ◽

Cited By ~ 3

Author(s):

Harvey F. Lodish

Keyword(s):

Conflicts Of Interest ◽

Inflammatory Diseases ◽

Stress Hormones ◽

Red Cells ◽

Red Cell ◽

Short Term ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Production ◽

Multiple Levels

Abstract SCI-1 Hematopoietic stem cells (HSC) undergo self-renewal and also generate all types of blood cells, including red cells, myeloid cells, and all immune system cells. Formation of red cells from HSC involves multiple cellular stages, including early erythroid-specific progenitors (burst-forming unit-erythroid [BFU-E]) that respond to several growth factors and an erythropoietin (Epo)-responsive progenitor, and the colony-forming unit-erythroid (CFU-E) that in 3 days generates ∼30 reticulocytes. In adult animals most CFU-E cells undergo apoptosis. A short-term need for red cells is met by increased Epo production by the kidney, which rescues increasing numbers of CFU-Es from apoptosis and increases red cell production over a few days. Chronic stress, such as in certain types of anemias and inflammatory diseases, leads to a marked increase in numbers of HSC and many types of progenitor cells. I will focus most of my talk on stress-triggered BFU-E proliferation and formation of CFU-Es, since our very recent work showed how glucocorticoids – an important class of stress hormones – as well as HIF-1α stimulate red cell production. Acting synergistically these allow ∼300 times more CFU-Es and erythroblasts to be formed from each BFU-E. More specifically, these molecules stimulate limited self-renewal of BFU-Es during cell division, thus maintaining progenitor immaturity and allowing over time increased numbers of CFU-E cells and thus mature red cells to be formed. This explains why certain corticosteroids are useful in treating some non-Epo responsive anemias, and I will present a molecular dissection of glucocorticoid and HIF-1α action on BFU-E progenitors that has led to the identification of genes critical for self-renewal and that suggest other possible avenues for treatment of chronic anemias. Disclosures: No relevant conflicts of interest to declare.

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DNMT3A Mutation Leads to Hematopoietic Dysregulation.

Blood ◽

10.1182/blood.v120.21.2382.2382 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2382-2382

Author(s):

Jie Xu ◽

Wei-na Zhang ◽

Tao Zhen ◽

Yang Li ◽

Jing-yi Shi ◽

...

Keyword(s):

Transcriptional Activation ◽

Dna Methyltransferase ◽

De Novo ◽

Conflicts Of Interest ◽

Epigenetic Modification ◽

Hematopoietic Cells ◽

In Vitro Assays ◽

Clinical Samples ◽

Hematopoietic Stem

Abstract Abstract 2382 Epigenetic modification process is required for the development of hematopoietic cells. DNA methyltransferase DNMT3A, responsible for de novo DNA methylation, was newly reported to have a high frequency of mutations in hematopoietic malignancies. Conditional knock-out of DNMT3A promoted self-renewal activity of murine hematopoietic stem cells (HSCs). However, the role of mutated DNMT3A in hematopoiesis and its regulative mechanism of epigenetic network mostly remain unknown. Here we showed that the Arg882His (R882H) hotspot locus on DNMT3A impaired the normal function of this enzyme and resulted in an abnormal increase of primitive hematopoietic cells. In both controlled in vivo and in vitro assays, we found that the cells transfected by R882H mutant promoted cell proliferation, while decreased the differentiation of myeloid lineage compared to those with wild type. Analysis of bone marrow (BM) cells from mice transduced by R882H reveals an expansion of Lin−Sca-1+C-kit+ populations and a reduction of mature myeloid cells. Meanwhile, a cluster of upregulated genes and downregulated lineage-specific differentiation genes associated with hematopoiesis were discovered in mice BM cells with R882H mutation. We further evaluated the association of mutated DNMT3A and HOXB4 which was previously detected to be highly expressed in clinical samples carrying R882 mutation. Compared with wildtype DNMT3A, R882H mutation disrupted the repression of HOXB4 by largely recruiting tri-methylated histone 3 lysine 4 (H3K4). Taken together, our results showed that R882H mutation disturbed HSC activity through H3K4 tri-methylation, and transcriptional activation of HSC-related genes. Disclosures: No relevant conflicts of interest to declare.

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Developmental Control of Polycomb Subunit Composition Mediates a Switch to Non-Canonical Functions during Hematopoiesis

Blood ◽

10.1182/blood.v124.21.241.241 ◽

2014 ◽

Vol 124 (21) ◽

pp. 241-241

Author(s):

Jian Xu ◽

Zhen Shao ◽

Dan Li ◽

Huafeng Xie ◽

Woojin Kim ◽

...

Keyword(s):

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Specific Activity ◽

Function Analysis ◽

Subunit Composition ◽

Erythroid Cells ◽

Developmental Control ◽

Hematopoietic Stem ◽

Enhancer Element ◽

Hematopoietic Malignancies

Abstract The epigenetic machinery plays crucial roles in hematopoiesis, and its deregulation drives the pathogenesis of blood disorders. Polycomb Repressive Complex 2 (PRC2) is a major class of epigenetic repressor that catalyzes the di/tri-methylation of histone H3 lysine 27 (or H3K27me2/3). The canonical PRC2 complex consists of EED, SUZ12, and the histone methyltransferase EZH2. The functions of PRC2 in hematopoiesis remain elusive due in large to the existence of two highly related enzymatic subunits EZH1 and EZH2. While amplification or overexpression of PRC2 proteins is common in many cancers, inactivating mutation in PRC2 is frequently found in hematopoietic malignancies, indicating that PRC2 can be oncogenic or tumor suppressive in different cellular contexts. In light of recent efforts to therapeutically target EZH2 enzyme activities or canonical EZH2-PRC2 functions in various hematopoietic malignancies, it will be critical to fully assess the context-specific activity of this epigenetic complex in normal and malignant developmental processes. The molecular mechanisms by which PRC2 regulates normal and neoplastic hematopoiesis is unclear, as are the non-redundant effects of canonical versus non-canonical PRC2 functions, which are mediated by EZH1 or EZH2 independent of H3K27me2/3. In this study, we demonstrate that the PRC2 enzymatic subunits EZH1 and EZH2 undergo an expression switch during hematopoiesis. EZH2 is highly expressed in primary human CD34+ hematopoietic stem/progenitor (HSPC) cells and progressively downregulated during erythroid and lymphoid specification, whereas EZH1 is significantly upregulated during differentiation. We next examined the in vivo stoichiometry of the PRC2 complexes by quantitative proteomics and revealed the existence of an EZH1-SUZ12 sub-complex lacking EED subunit in human erythroid cells. Through genome scale chromatin occupancy (by ChIP-seq) and transcriptional profiling (by RNA-seq) analyses, we provide evidence that EZH1 together with SUZ12 form a non-canonical PRC2 complex, occupy active chromatin domains marked by H3K4me3 and H3K27me1, and positively regulate gene expression. Furthermore, loss of EZH2 expression leads to global repositioning of EZH1 chromatin occupancy to EZH2 targets, and EZH1 complements EZH2 loss within canonical PRC2 target genes. To elucidate the regulatory networks underlying the developmental control of EZH1/2 switch, we profiled the histone modifications and chromatin accessibility surrounding the EZH1 gene in both CD34+ HSPCs and committed erythroid cells. We identified and characterized an erythroid-selective enhancer element that is indispensable for the transcriptional activation of EZH1. Loss of function analysis using CRISPR/cas9-mediated enhancer deletion results in markedly decrease in EZH1 expression in human erythroid cells. Moreover, a switch from GATA2 to GATA1 expression controls the developmental EZH1/2 switch by differential association with distinct EZH1 enhancers during erythroid differentiation. Thus, the lineage- and developmental stage-specific regulation of PRC2 subunit composition leads to a switch from canonical silencing to non-canonical PRC2 functions. Our study also establishes a molecular link between the switch of master lineage regulators and developmental control of PRC2 composition, providing a means to coordinate linage-specific transcription and accompanying changes in the epigenetic landscape during blood stem cell specification. Disclosures No relevant conflicts of interest to declare.

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COL4A1 is a Novel Causative Gene Responsible for Congenital Hemolytic Anemia, Representing Characteristic Clinical Course in Infants

Blood ◽

10.1182/blood.v126.23.934.934 ◽

2015 ◽

Vol 126 (23) ◽

pp. 934-934

Author(s):

Hiromi Ogura ◽

Shouichi Ohga ◽

Takako Aoki ◽

Taiju Utsugisawa ◽

Hidehiro Takahashi ◽

...

Keyword(s):

Hemolytic Anemia ◽

De Novo ◽

Conflicts Of Interest ◽

Small Vessel Disease ◽

Gene Mutations ◽

Basement Membranes ◽

Red Cell ◽

Missense Mutations ◽

Causative Gene ◽

Congenital Hemolytic Anemia

Abstract We have been working on the differential diagnosis of congenital hemolytic anemia, but even with extensive analysis of hemoglobin, red cell membrane and enzymes, approximately 40% of patients remained to be diagnosed. In this study, we analyzed 17 undiagnosed hemolytic anemia subjects under the age of 1 by whole-exome sequencing, and identified COL4A1 gene mutations in 5 cases (29.4%). All patients were de novo cases without family histories and exhibited moderate to severe neonatal hemolytic anemia: Hgb, 5.2-9.3 g/dl; MCV, 90.0-126.9; MCHC, 29.9-32.7; and reticulocyte count, 9.2-33.0%. Either schizocytes or poikilocytes were observed in peripheral blood smears of 3 cases, suggesting that the microangiopathy was attributable to hemolysis. Previous reports showed that mutation of COL4A1 accounts for brain small-vessel disease characterized by stroke and eye abnormalities and the most characteristic complications of the present cases were congenital anomaly in the central nervous system, such as porencephaly, schizencephaly, congenital hydrocephalus, cataracts or paraventricular calcification, as reported previously. Hemolytic anemia became less severe within 2 months after birth, and all cases no longer required red cell transfusion after Day 50. COL4A1 encodes subtype 1 of type IV collagen, which is most abundantly expressed in basement membranes, including the vasculature. The COL4A1 gene mutations identified in the cases were all novel missense mutations except one, located in exons 26, 27, 37, 38 and 51. Although the pathophysiological significance of the mutations remains unclear, COL4A1 is the first identified causative gene responsible for congenital hemolytic anemia without intrinsic defects of red blood cells, and mutation of COL4A1 is the most prevalent cause of neonatal hemolytic anemia. Disclosures No relevant conflicts of interest to declare.

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Elevated Red Cell Reduced Glutathione Is a Novel Biomarker of Diamond-Blackfan Anemia

Blood ◽

10.1182/blood.v124.21.1342.1342 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1342-1342

Author(s):

Taiju Utsugisawa ◽

Toshitaka Uchiyama ◽

Hiromi Ogura ◽

Takako Aoki ◽

Isao Hamaguchi ◽

...

Keyword(s):

Conflicts Of Interest ◽

Reduced Glutathione ◽

Recent Observation ◽

Positive Family History ◽

Gene Mutations ◽

Support Vector ◽

Red Cell ◽

Erythroid Cells ◽

Linear Discriminant ◽

Diamond Blackfan Anemia

Abstract Diamond-Blackfan anemia (DBA) is a rare congenital red cell aplasia characterized by congenital anomalies and predisposition to cancer. Recent observation disclosed that heterogeneous mutations in ribosomal protein (RP) genes are present in approximately 50% of patients, suggesting that diagnosis should be made by clinical phenotypes such as age, hematological findings or positive family history. Although elevated activity of red cell adenosine deaminase (eADA) has been utilized as a useful biomarker for differential diagnosis of DBA, approximately 20% of DBA patients are eADA-negative. Recent observations suggested that ribosomal haploinsufficiency increases oxidative stress, leading to p53 gene activation and premature death of erythroid cells. We hypothesized that reduced glutathione (GSH), an essential antioxidant of erythroid cells, might be upregulated in red cells of DBA subjects. In order to test this hypothesis, we examined red cell GSH as well as eADA of 22 patients in 18 DBA families, in whom we had identified gene mutations in RPS19, RPL5, RPL11, RPS10, RPS17 or RPS35a. All except one DBA patients showed elevated GSH (>88.6 mg/dl RBC, M+SD), whereas 17 out of 22 patients exhibited elevated eADA (>2.31 IU/g Hb, M+3SD). We also examined 14 unaffected members of the DBA families, with 1 out of 14 subjects showing elevated GSH and none showing elevated eADA. We performed linear discriminant analysis between DBA and non-DBA subjects with both eADA and GSH using the Support Vector Machine (SVM) from 36 subjects, and successfully obtained a formula to discriminate DBA from unaffected subjects: 0.937*eADA+0.0702*GSH-7.9044 >0. By using this formula, all DBA examined can be diagnosed and unaffected family members can be excluded. Since approximately 50% of clinically diagnosed DBA cases have no causative RP gene mutations, the combined assessment of eADA and GSH might be quite useful for biochemical diagnosis of DBA. Disclosures No relevant conflicts of interest to declare.

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