scholarly journals Functional Overlap between the SEC23 Paralogs Suggests a Novel Treatment Paradigm for Congenital Dyserythropoietic Anemia Type II

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2221-2221
Author(s):  
Richard King ◽  
Ann Friedman ◽  
Zesen Lin ◽  
Rami Khoriaty
Keyword(s):  
Amino Acids ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Type Ii ◽  
Erythroid Cells ◽  
Loss Of Function ◽  
Congenital Dyserythropoietic Anemia ◽  
Erythroid Precursors ◽  
Copii Vesicles ◽  
Wildtype Littermate

Congenital dyserythropoietic anemia type II (CDAII), an autosomal recessive disease characterized by ineffective erythropoiesis and increased percentage of bi-nucleated erythroid precursors in the bone marrow (BM), results from loss of function mutations in SEC23B, which encodes a core component of COPII vesicles. Approximately 8,000 secretory proteins are transported from the endoplasmic reticulum to the Golgi apparatus via COPII vesicles, suggesting that a defect in this pathway would result in a profound systemic phenotype. However, CDAII patients exhibit a specific erythroid phenotype, with no other defects described. Mammals have 2 paralogs for SEC23, SEC23A and SEC23B. In contrast to SEC23B mutations, bi-allelic SEC23A loss of function mutations in humans result in cranio-lenticulo-sutural dysplasia, a disease characterized by skeletal defect but normal erythropoiesis. We previously demonstrated that a SEC23B-A chimeric protein composed of the first 122 amino acids of SEC23B followed by amino acids 123-765 of SEC23A overlaps in function with SEC23B, suggesting that the 2 SEC23 paralogs are functionally interchangeable. However, to rule out the possibility that the functional overlap was due to the first 122 amino acids of SEC23B, we generated a bacterial artificial chromosome (BAC) transgene that expresses the full Sec23a coding sequence from the endogenous genomic locus of Sec23b (Sec23b-a BAC). We crossed the Sec23b-a BAC to the Sec23b null allele (Sec23b-) and demonstrated that this BAC rescues the phenotype of mice deficient in Sec23b (Sec23b-/-). Therefore, we now conclusively demonstrate that the SEC23A protein functionally replaces SEC23B when expressed from the endogenous regulatory elements of Sec23b. We have previously shown that mice with erythroid-specific and pan-hematopoietic SEC23B deficiency exhibit a normal erythroid phenotype. In light of the functional overlap between SEC23A and SEC23B, we hypothesized that mice with erythroid-specific deficiency for SEC23A, alone or in combination with SEC23B, might exhibit an erythroid phenotype. First, we generated mice with erythroid-specific (EpoR-Cre) SEC23A deficiency. These mice were observed at the expected Mendelian ratios at weaning. Complete (or near complete) excision of the Sec23a floxed (Sec23afl) allele was confirmed in the erythroid cells. Peripheral blood counts, BM cellularity and morphology, and percent and distribution of BM erythroid cells among the 5 stages of maturation were indistinguishable between mice with erythroid SEC23A deficiency and wildtype littermate controls. Additionally, the percentage of bi-nucleated erythroid precursors were not increased in Sec23afl/flEpoR-Cre+ mice. Thus, mice with erythroid-specific SEC23A deficiency do not exhibit an erythroid phenotype. Similarly, mice with pan-hematopoietic SEC23A deficiency (Vav1-Cre) do not exhibit a hematologic phenotype. Next, we generated mice with Sec23a deletion and Sec23b haploinsufficiency in the erythroid compartments. These mice exhibited normal survival, a mild reduction in hemoglobin levels (p = 0.014), and a block in late erythroid maturation (Stage V erythroid cells were reduced to 22.6% compared to 30.3% in control mice; p=0.08). In contrast, mice with erythroid-specific deletion for all 4 Sec23 alleles (combined SEC23A/B deficiency) died at mid-embryogenesis exhibiting reduced size and appearing pale compared to wildtype littermate controls, with histologic evidence of dyserythropoiesis reminiscent of human CDAII. Overall, these results suggest a requirement for a threshold level of total SEC23 (combined SEC23A/B) expression in the erythroid compartment. These results also suggest that the defect in CDAII is intrinsic to the RBC. Finally, we generated K562 cells with either SEC23B or SEC23A deletion using CRISPR/Cas9 genome editing. SEC23B or SEC23A deletion alone was tolerated in the K562 cells. However, combined deletion of SEC23A and SEC23B was not tolerated. Taken together, the results summarized above demonstrate that SEC23A and SEC23B appear to compensate for one another's function in murine and human erythroid cells. This finding suggests a potential therapeutic role for increasing expression of SEC23A to compensate for SEC23B deficiency in CDAII. This work is currently ongoing. Disclosures No relevant conflicts of interest to declare.

scholarly journals RAP-011 Rescues the Disease Phenotype in a Cellular Model of Congenital Dyserythropoietic Anemia Type II by Inhibiting the SMAD2-3 Pathway

2020 ◽  
Vol 21 (15) ◽  
pp. 5577
Author(s):  
Gianluca De Rosa ◽  
Immacolata Andolfo ◽  
Roberta Marra ◽  
Francesco Manna ◽  
Barbara Eleni Rosato ◽  
...  
Keyword(s):  
Gene Expression ◽  
K562 Cells ◽  
Type Ii ◽  
Disease Phenotype ◽  
Loss Of Function ◽  
Ineffective Erythropoiesis ◽  
Differentiation Markers ◽  
Hematopoietic Stem ◽  
Congenital Dyserythropoietic Anemia ◽  
Cda Ii

Congenital dyserythropoietic anemia type II (CDA II) is a hypo-productive anemia defined by ineffective erythropoiesis through maturation arrest of erythroid precursors. CDA II is an autosomal recessive disorder due to loss-of-function mutations in SEC23B. Currently, management of patients with CDA II is based on transfusions, splenectomy, or hematopoietic stem-cell transplantation. Several studies have highlighted benefits of ACE-011 (sotatercept) treatment of ineffective erythropoiesis, which acts as a ligand trap against growth differentiation factor (GDF)11. Herein, we show that GDF11 levels are increased in CDA II, which suggests sotatercept as a targeted therapy for treatment of these patients. Treatment of stable clones of SEC23B-silenced erythroleukemia K562 cells with the iron-containing porphyrin hemin plus GDF11 increased expression of pSMAD2 and reduced nuclear localization of the transcription factor GATA1, with subsequent reduced gene expression of erythroid differentiation markers. We demonstrate that treatment of these SEC23B-silenced K562 cells with RAP-011, a “murinized” ortholog of sotatercept, rescues the disease phenotype by restoring gene expression of erythroid markers through inhibition of the phosphorylated SMAD2 pathway. Our data also demonstrate the effect of RAP-011 treatment in reducing the expression of erythroferrone in vitro, thus suggesting a possible beneficial role of the use of sotatercept in the management of iron overload in patients with CDA II.

Overlap of SEC23A and SEC23B Function Suggests a Novel Therapeutic Approach for Congenital Dyserythropoietic Anemia Type II

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 80-80
Author(s):  
Rami Khoriaty ◽  
Angela Weyand ◽  
Geoffrey Hesketh ◽  
Amélie Bernard ◽  
Lesley Everett ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Genetically Engineered ◽  
Hek293 Cells ◽  
Secretory Proteins ◽  
Type Ii ◽  
Loss Of Function ◽  
Genomic Locus ◽  
Cell Stage ◽  
Congenital Dyserythropoietic Anemia ◽  
Copii Vesicles

Abstract Congenital Dyserythropoietic Anemia type II (CDAII) is an autosomal recessive disease characterized by anemia and increased bone marrow (BM) bi/multi-nucleated erythroblasts. CDAII results from loss of function mutations in SEC23B encoding a core component of coat complex protein II (COPII) vesicles, which transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Despite the identification of the genetic cause of CDAII, the pathophysiology of the disease remains unknown. Morpholino-induced SEC23B deficiency in zebrafish (ZF) has been previously reported to result in an erythroid phenotype mimicking CDAII (Shwartz et al, Nature genetics 2009), suggesting conservation of the underlying CDAII mechanism from fish to humans. Thus, we were puzzled to observe the absence of anemia or other CDAII characteristics in mice with erythroid specific (EpoR -Cre) and pan-hematopoietic (Vav1 -Cre) SEC23B deficiency (Khoriaty et al, MBC and Khoriaty et al, Sci Rep). To re-examine the ZF phenotype, we injected the morpholino targeting Sec23b into one-cell stage ZF embryos demonstrating no increase in circulating bi-nucleated erythroid cells, in contrast to the previous report. Given the variable knock-down that can result from morpholinos, we next generated ZF heterozygous for a 53 bp deletion (Sec23b+/-) using CRISPR/Cas9 genome editing. Intercrosses between Sec23b+/- ZF demonstrated lethality of Sec23b-/- ZF between days 17-21. However, the percentage of circulating bi-nucleated erythrocytes observed at day 16 was indistinguishable between Sec23b-/- ZF and wildtype (WT) clutch mate controls. Mammals and fish express two paralogs for SEC23, SEC23A and SEC23B, encoding highly related (~85%) proteins. To investigate the different functions of SEC23A and SEC23B, we defined the SEC23A and SEC23B interactomes using "BioID" (proximity dependent biotinylation) in HEK293 cells expressing BirA*-tagged SEC23A, SEC23B, or GFP control. Surprisingly, SEC23A and SEC23B exhibit indistinguishable interactomes. We also demonstrated that both mouse and human SEC23 paralogs can complement SEC23 deficiency in yeast. Similarly, rescue of the Sec23b-/- lethal phenotype in ZF by a Sec23a transgene demonstrated at least partial functional overlap of SEC23A/SEC23B function in vertebrates. To extend these observations to mammals, we genetically engineered the murine Sec23a cDNA into the endogenous mouse genomic locus of Sec23b . We demonstrated that SEC23B-deficient mice (previously shown to die perinatally from pancreatic degeneration) are rescued by SEC23A, exhibiting normal survival and pancreas histology, with no abnormalities apparent on detailed hematologic and anatomic examination. The expression of SEC23A and SEC23B mRNAs in human and mouse BMs were examined by qRT-PCR. SEC23B is the predominantly expressed paralog in human BM, with greater levels of SEC23A and reduced SEC23B in mouse BM. We therefore hypothesized that mice with erythroid deficiency of SEC23A alone or combined SEC23A/SEC23B deficiency might exhibit an erythroid defect. We first generated mice with erythroid-specific SEC23A deficiency, with the latter mice exhibiting no anemia or other CDAII characteristic. In contrast, mice with combined erythroid SEC23A and SEC23B deficiency die at ~E12.5, exhibiting reduced size and appear white in color compared to their WT litter mate controls, consistent with requirement of SEC23 in the erythroid compartment. Taken together, these data suggest complete (or near complete) overlap in function between SEC23A and SEC23B, and suggest that therapies that increase the expression of either SEC23 paralog might prove effective in treating CDAII. This paradigm might also apply to other disorders due to mutations in paralogous genes. Finally, our findings also suggest that a switch in paralog expression could account for other disparate disease phenotypes observed between animal models and humans. Disclosures No relevant conflicts of interest to declare.

Chromatin Conformation and a Distal Regulatory Element Activate the Human Erythroid Ankyrin-1 Promoter.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 803-803
Author(s):  
Ashley N. Owen ◽  
Robert I. Liem ◽  
Andre M. Pilon ◽  
Patrick G. Gallagher ◽  
David M. Bodine
Keyword(s):  
Reporter Gene ◽  
Regulatory Element ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Dnase I ◽  
Luciferase Reporter ◽  
Erythroid Cells ◽  
Luciferase Reporter Gene ◽  
Human And Mouse ◽  
In Erythroid Cells

Abstract Ankyrin forms the bridge between the spectrin/actin network of the erythrocyte membrane skeleton and the red cell membrane by binding to both β-spectrin and band 3. The erythrocyte ankyrin promoter (Ank-1E) is active only in erythroid cells, while two other Ank-1 promoters located 20 kb downstream and 40 kb upstream of Ank-1E are active in the cerebellum and muscle cells respectively. We have been studying the mechanism by which the Ank-1E promoter becomes active in erythroid cells by studying the cis acting regulatory elements and the chromatin structure of the Ank-1 promoter region. We have previously shown that the sequences between −296 and −15 of the Ank-1E promoter are fully sufficient for erythroid specific, copy number dependent uniform expression of reporter genes in transgenic mice. We have also mapped a DNase I Hypersensitive site (5′HS) between −300 and −100 of the human and mouse Ank-1E promoters in human K562 and mouse fetal liver cells. Both the mouse and human 5′HS are capable of preventing the silencing of a β-globin/GFP reporter gene in K562 cells, establishing that they function as barrier elements. Consistent with this observation, the human and mouse 5′HS are hyperacetylated in erythroid cells. The chromatin 10 kb 5′ to the 5′HS is DNase I resistant (associated with inactive chromatin) in human and mouse erythroid and non-erythroid cells. Approximately 6 kb 3′ to 5′HS are two adjacent HS (3′HS1, 3′HS2). Beyond 3′HS2 the chromatin is also DNase I resistant in both human and mouse erythroid and non-erythroid cells. Between 5′HS and 3′HS1 the 6kb region is DNase I sensitive (active) in erythroid cells but not in other cell types. We hypothesized that this 6 kb region contains regulatory elements that activate the Ank-1E promoter. To screen for regulatory elements we isolated overlapping segments of a 10 kb region extending from 2 kb upstream of 5′HS to 2 kb downstream of 3′HS2. We inserted these fragments into a plasmid vector containing the Ank-1E promoter linked to a luciferase reporter gene and transfected these constructs into K562 cells. A single region up regulated Ank-1E/luciferase expression. This region mapped to a 211bp segment that included 3′HS1, but did not include 3′HS2. A fragment containing only 3′HS2 did not up regulate an Ank-1E/luciferase reporter gene, but 3′HS2 was capable of preventing the silencing of a β-globin/Green Fluorescent Protein reporter gene in K562 cells, demonstrating barrier activity. The region around 3′HS1 and 2 was also a site of histone hyperacetylation. The sequence of the 211 bp fragment containing 3′HS1 does not contain consensus sequences for any known erythroid-specific transcription factors, but does contain potential binding sites fro Sp1, AP-1 and E-box binding proteins. Using the Chromatin Conformation Capture assay we demonstrated that 5′HS and 3′HS1 and 2 are in close proximity in K562 chromatin, but are not closely associated in chromatin from other cell types. We propose that an erythroid-specific chromatin loop brings 3′HS1 and 2 into proximity with 5′HS, adjacent to the Ank-1E promoter. This interaction translocates the positive regulatory element in 3′HS1 to the Ank-1E promoter allowing the Ank-1E promoter to become active in erythroid cells.

Congenital Dyserythropoietic Anemia Type II: Excessive Endoplasmic Reticulum in Erythroid Cells and Mitochondrial Inclusions of Hepatic Cells

1971 ◽  
Vol 29 ◽  
pp. 294-295
Author(s):  
George Hug ◽  
K. Y. Wong ◽  
Beatrice Lampkin
Keyword(s):  
Bone Marrow ◽  
Human Serum ◽  
Present Report ◽  
Initial Study ◽  
Red Cells ◽  
Type I ◽  
Red Cell ◽  
Type Ii ◽  
Erythroid Cells ◽  
Congenital Dyserythropoietic Anemia

Congenital dyserythropoietic anemia (CDA) as described in 1966 was characterized by (i) erythroblastic multinuclearity and (ii) lysis of the patient's red cells in acidified compatible normal human serum. This condition has since been labeled CDA Type II to distinguish it from a similar entity, CDA Type I, with erythroblastic multinuclearity but without red cell lysis in acidified human serum. According to this classification, our initial study of bone marrow ultrastructure in CDA concerned a girl with Type II. Her bone marrow contained erythroid cells with excessive cytoplasmic membranes and multiple nuclei. The present report illustrates this observation. The patient was a 12 year old white girl with congenital anemia and benign recurrent jaundice. Hemolysis was not present since Cr51 red cell survival time was normal. Bone marrow aspirates (Figure 1, 2 and 4) circulating red cells (Figure 3) and hepatic biopsy specimens were examined. The markers indicate 0.5 microns and N designates nucleus. The myeloid series was normal. Figure 1 shows a representative polychromatophilic normoblast.

scholarly journals A common SEC23B missense mutation in congenital dyserythropoietic anemia type II leads to growth restriction and symptoms of chronic pancreatitis in mice

2021 ◽  
Author(s):  
Wei Wei ◽  
Zhigang Liu ◽  
Chao Zhang ◽  
Rami Khoriaty ◽  
Min Zhu ◽  
...  
Keyword(s):  
Chronic Pancreatitis ◽  
Missense Mutation ◽  
Growth Retardation ◽  
Perinatal Death ◽  
Interstitial Fibrosis ◽  
Critical Role ◽  
Type Ii ◽  
Loss Of Function ◽  
Exocrine Insufficiency ◽  
Congenital Dyserythropoietic Anemia

In humans, loss-of-function mutations in SEC23B result in congenital dyserythropoietic anemia type II (CDAII). Complete deficiency of Sec23b in mice leads to perinatal death caused by massive degeneration of professional secretory tissues with no CDAII phenotype. Functions of SEC23B in postnatal mice are unclear. In this study, we generated mice with the E109K mutation (Sec23bki/ki), the most common CDAII missense mutation. The E109K mutation leads to decreased SEC23B protein level, and results in mislocalization of SEC23B. However, Sec23bki/ki mice showed no obvious abnormalities. Hemizygous (Sec23bki/ko) mice exhibit a partial lethal phenotype, with half of these mice survive past weaning. Sec23bki/ko mice had chronic pancreatic histology changes such as interstitial fibrosis, white blood cell infiltration and exocrine insufficiency. Increased ER stress was found in Sec23bki/ko pancreas, associated with increased apoptosis. Moreover, Sec23bki/ko mice exhibited severe growth retardation accompanied by growth hormone (GH) insensitivity, reminiscent of the Laron syndrome. Interestingly, mice with hepatocyte-specific Sec23b deletion grow normally, suggesting non-liver origin of the phenotype. Inflammation associated with chronic pancreatic deficiency may explain GH insensitivity in Sec23bki/ko mice. Our results indicate that phenotype severities are linked to the residual functions of SEC23B in mice, further demonstrating a critical role of SEC23B in pancreatic acinar function in adult mice. The Sec23bki/ko mice provide a novel model of chronic pancreatitis and growth retardation.

scholarly journals Unbalanced Globin Chain Synthesis in Congenital Dyserythropoietic Anemia

Blood ◽  
1973 ◽  
Vol 42 (6) ◽  
pp. 843-850 ◽  
Author(s):  
Marilyn A. Hruby ◽  
R. George Mason ◽  
George R. Honig
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Type Ii ◽  
Erythroid Cells ◽  
Globin Chain ◽  
Congenital Dyserythropoietic Anemia ◽  
Thalassemia Trait ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

Abstract Hematologic evaluation of a 5-yr-old girl with lifelong anemia demonstrated the characteristic findings of congenital dyserythropoietic anemia (CDA) type II. Globin chain synthesis was studied in vitro by measuring the incorporation of L-leucine-14C into globin by peripheral blood and bone marrow erythroid cells. In cells from the child and from both of her parents an abnormal balance between the synthesis of the α and non-α globin components of hemoglobin was observed, the α chains being synthesized in excess. Neither parent demonstrated microcytosis, hypochromia, or other findings suggestive of β-thalassemia trait.

scholarly journals Identification of a novel homozygous loss-of-function mutation in FUCA1 gene causing severe fucosidosis: A case report

2021 ◽  
Vol 49 (4) ◽  
pp. 030006052110059
Author(s):  
Xinwen Zhang ◽  
Shaozhi Zhao ◽  
Hongwei Liu ◽  
Xiaoyan Wang ◽  
Xiaolei Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Lysosomal Storage Disorder ◽  
Autosomal Recessive ◽  
Signs And Symptoms ◽  
Lysosomal Storage ◽  
Loss Of Function ◽  
Wild Type ◽  
Single Nucleotide ◽  
Whole Exome ◽  
Exon 1

Fucosidosis is a rare lysosomal storage disorder characterized by deficiency of α-L-fucosidase with an autosomal recessive mode of inheritance. Here, we describe a 4-year-old Chinese boy with signs and symptoms of fucosidosis but his parents were phenotypically normal. Whole exome sequencing (WES) identified a novel homozygous single nucleotide deletion (c.82delG) in the exon 1 of the FUCA1 gene. This mutation will lead to a frameshift which will result in the formation of a truncated FUCA1 protein (p.Val28Cysfs*105) of 132 amino acids approximately one-third the size of the wild type FUCA1 protein (466 amino acids). Both parents were carrying the mutation in a heterozygous state. This study expands the mutational spectrum of the FUCA1 gene associated with fucosidosis and emphasises the benefits of WES for accurate and timely clinical diagnosis of this rare disease.

scholarly journals GATA1 erythroid-specific regulation of SEC23B expression and its implication in the pathogenesis of congenital dyserythropoietic anemia type II

Haematologica ◽  
2017 ◽  
Vol 102 (9) ◽  
pp. e371-e374 ◽  
Author(s):  
Roberta Russo ◽  
Immacolata Andolfo ◽  
Antonella Gambale ◽  
Gianluca De Rosa ◽  
Francesco Manna ◽  
...  
Keyword(s):  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
Specific Regulation
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