Congenital Dyserythropoietic Anemia Type II Research Articles

Whole-Genome CRISPR-Cas9 Screen Identifies ZBTB7A As a Potential Therapeutic Target for Cda-II

Congenital dyserythropoietic anemia type II (CDA-II) is an autosomal recessive disease characterized by anemia, ineffective erythropoiesis, and increased bone marrow bi-nucleated erythroblasts. CDA-II is caused by loss-of-function mutations in SEC23B, which encodes a component of coat protein complex II (COP-II) vesicles/tubules that transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Mammals express two SEC23 paralogs, SEC23A and SEC23B. We have previously shown that SEC23A is functionally interchangeable with SEC23B and that increased expression of SEC23A rescues the SEC23B-deficient erythroid differentiation defect observed in CDA-II. Here, we utilized our recently generated clonal SEC23B deficient HUDEP-2 cell line to identify novel therapeutic targets for CDAII. SEC23B deficient HUDEP2 cells survive and grow normally when cultured as pro-erythroblasts in ‘maintenance media’. However, upon culturing these cells in ‘differentiation media’, which results in semi-synchronous erythroid differentiation, differentiated SEC23B-null HUDEP2 cells exhibit reduced growth, impaired differentiation, increased bi-nucleated erythroid cells, and erythroid cell death, features of CDA-II. To identify novel genes that play important roles in the pathophysiology of CDAII, we developed a functional genome-scale CRISPR knockout screen to define genes that when deleted, rescue the SEC23B-null erythroid differentiation defect. SEC23B null HUDEP2 cells cultured in maintenance media were infected with the hGeCKO-v2 lentiviral library at a multiplicity of infection (MOI) of ~0.3, allowing most transduced cells to receive 1 sgRNA (and Cas9) to knockout 1 gene only. Transduced cells were puromycin selected and passaged in expansion media for 14 days. SEC23B-null HUDEP2 cells were then differentiated for 10 days. Cells were harvested prior to differentiation (Day 0, D0), and differentiated (orthochromatic) cells were sorted at D10 of differentiation. Integrated sgRNAs were quantified by deep sequencing. sgRNAs targeting ZBTB7A were amongst the most enriched in D10 compared to D0 erythroid cells. To validate these findings, we transduced SEC23B-null HUDEP2 cells with two independent and efficient ZBTB7A-targeting sgRNAs. We found that ZBTB7A deletion (using either of the 2 sgRNAs) rescued the lethality and differentiation defect of SEC23B null HUDEP2 cells undergoing differentiation. We next generated 7 clonal cell lines with combined deletion for ZBTB7A and SEC23B and confirmed that these cells exhibited normal growth and differentiation indistinguishable from wildtype HUDEP2 cells. The rescuing effect of targeting ZBTB7A was also validated in SEC23B mutated human CD34+ hematopoietic stem and progenitor cell culture. Since SEC23A can functionally replace SEC23B in erythroid cells, we quantified the SEC23A mRNA level in SEC23B-null HUDEP2 cells deleted for ZBTB7A, by qRT-PCR. In early preliminary results, deletion of ZBTB7A resulted in a profound (~10 fold) increase in SEC23A mRNA expression, to a level predicted to be sufficient to rescue the SEC23B null erythroid differentiation defect. ChIP-seq analysis demonstrated that ZBTB7A occupies the SEC23A promoter in HUDEP2 cells. Taken together, these data suggest that ZBTB7A represses SEC23A expression during erythroid maturation and that in the setting of ZBTB7A deletion, SEC23A expression is de-repressed, resulting in rescue of the CDA-II erythroid differentiation defect. Genome editing of the ZBTB7A binding sites in the SEC23A promoter is ongoing in SEC23B-null HUDEP-2 cells. We will determine the impact of the latter editing on SEC23A expression and erythroid differentiation.

A Novel Beta Globin Frameshift Mutation Causing Autosomal Dominant Beta Thalassemia

Background: Dominantly inherited β-Thalassemia (β-thal) is a rare condition that results from a heterozygous mutation in the β-globin gene ( HBB), leading to the synthesis of an unstable β-globin chain, most frequently an elongated protein. Although heterogeneous, it presents not as β-thalassemia trait but rather with a more severe phenotype, resembling β-thal major or intermedia, with chronic hemolytic anemia and splenomegaly. There are approximately 150 known dominantly inherited α- and β-globin gene mutations (http://globin.cse.psu.edu/). We present a case of a novel HBB mutation leading to a dominant form of β-thalassemia, outlining our approach to attaining an appropriate diagnosis and management leading to transfusion independence. Case: A 32-year-old Caucasian female was referred to our center with a diagnosis of congenital dyserythropoietic anemia type II (CDA-II), based on bone marrow studies at 12 years of age which had demonstrated a hypercellular marrow with erythroid hyperplasia and 10% binucleated red cells. Molecular testing had not been performed. Her history was negative for developmental delay, growth retardation, or family history of anemia. She had presented to medical attention at age 11 with symptomatic anemia. Prior treatments included steroids, IVIG, and erythropoietin, with no significant improvement of her anemia. She underwent splenectomy at 19 years of age due to the hemolytic component of her anemia with the clinical diagnosis of CDA-II, with a mild improvement in baseline hemoglobin from 70-80g/L to 80-90g/L. Spleen pathology demonstrated splenomegaly (weight of 1kg) and mild iron deposition. At the time of presentation at our centre, her predominant symptom was fatigue, for which she was initiated on a chronic transfusion protocol, to maintain a pretransfusion hemoglobin of 90 g/L; she received a total of 53 units from October 2018 to November 2022. She subsequently developed worsening iron overload (ferritin 888, TSAT 80%, and LIC of 8.1mg of iron per gram of liver dry weight) requiring initiation of iron chelation therapy. Given her presumptive diagnosis of CDA-II she was later enrolled in the Congenital Dyserythropoietic Anemia Registry (CDAR) for North America (https://www.clinicaltrials.gov/study/NCT02964494). Molecular testing by next generation sequencing (NGS) on a CDA gene-panel at that time failed to identify a causative CDA mutation. Further testing with whole exome sequencing revealed a one base-pair deletion in exon 2 of the β-globin gene, creating a frameshift variant at codon 96 and ending in a premature STOP, 63 codons downstream p.(Lys 96Asnfs*63) (Figure 1). This mutation led to an elongated β-globin chain and an unstable hemoglobinopathy predicted to be the cause of her presentation. Hemoglobinopathy screen revealed HBF 8.7%, HBA 87.5%, and HBA2 3.8%. Based on the above testing, a diagnosis of dominant β-thalassemia intermedia due to HBB NM_00518.4, c.288delG, p.K96fs, recognizing that she had been previously misdiagnosed with CDA-II. Based on the revised diagnosis, the patient was initiated on Luspatercept 1mg/kg SC q3 weeks, with rapid improvement in her pre-transfusion hemoglobin to >100g/L as well as in her symptomatology and quality of life (Figure 2), rendering her transfusion independent. Discussion: This case report, to our knowledge, demonstrates a novel β-globin gene mutation leading to dominant β-thalassemia, with the phenotype of β-thal-intermedia. The latency between presentation and diagnosis demonstrates the challenges associated with diagnosing rare hemolytic anemias and highlights the importance of molecular testing to confirm the diagnosis, especially before proceeding to splenectomy which may predispose to vasculopathies in some of these diseases, such as congenital dyserythropoietic anemia type I and unstable hemoglobinopathies.

Congenital Dyserythropoietic Anemia Type II: An Update from the Congenital Dyseryhtropoietic Anemia Registry of North America (CDAR)

Congenital Dyserythropoietic Anemias (CDAs) are a heterogeneous group of rare anemias characterized by ineffective erythropoiesis and hemolysis. The CDA registry in North America (CDAR; NCT02964494) was established to facilitate natural history studies. Participants who do not have a genetic diagnosis are offered family-trio whole genome sequencing (WGS) and elect to provide samples to CDAR biorepository to support collaborative mechanistic studies. CDA type II (CDA-II), the most common CDA type, is an autosomal recessive disease caused by biallelic SEC23Bvariants. CDA-II presents with hemolytic anemia with suboptimal reticulocytosis, iron overload, splenomegaly, and characteristic binucleation in 10-30% of the bone marrow (BM) erythroblasts. Although CDA-II is a recessive disorder, only one SEC23B variant is found in some patients. Here, we present the genetic and clinical data of CDA-II patients enrolled in CDAR so far. As of July 2023, 169 individuals (85 patients; 84 unaffected family members) enrolled in CDAR. Nine have a confirmed CDA-II diagnosis. Six patients have biallelic SEC23B pathogenic variants and 3 have only one variant identified. WGS for the 3 patients with a single variant revealed rare deep-intronic candidate variants. Since SEC23Bis ubiquitously expressed, we studied SEC23B protein levels in patient-derived lymphoblastoid cell lines generated for the CDAR biorepository. Patients with a single SEC23B variant exhibited an expression level comparable to those with biallelic mutations (Figure). The candidate deep intronic variants are currently being investigated for pathogenicity. Seven patients presented with hemolytic anemia during infancy, but the age of diagnosis was delayed by a mean of 12.5 yrs (0.1 to 55 y.o). Blood smears showed mild macrocytosis, poikilocytosis, and several spherocytes congruent with an osmotic gradient ektacytometry resembling mild hereditary spherocytosis. BM examination, when performed, exhibited erythroid hyperplasia, prominent dyserythropoiesis, and binucleation in ≥ 10% of erythroblasts. Anemia severity was variable: 6 patients were transfusion-dependent, 1 received intermittent transfusions, and 2 did not need blood transfusions. Two became transfusion-independent after splenectomy at ages 6 and 7 y.o, and two underwent successful matched-sibling BMT at age 10 and 26 y.o, after adequate chelation (liver iron concentration [LIC]<7 mg/g) Iron overload, disproportionate to transfusions, was present in 6 patients; 5 received iron chelation. All 5 patients with available MRI results had hepatic hemosiderosis (LIC range 3.4 to 21.8 mg/g) and evidence of myocardial hemosiderosis (T2* range 6.3 to 20 ms). Ferritin values were variable (range 58 to 1631.5 ng/ml) and discordant from LIC and T2* measurements. Notably, ferritin underestimated iron overload burden: 2 patients with normal ferritin (58 and 160 ng/mL) had hepatic hemosiderosis (LIC 6.1 and 3.4 mg/g respectively), and patients with severe hepatic and myocardial hemosiderosis (LIC 16.6-21.8 mg/g; T2* 6.3-7.8 ms) had modest ferritin values 1475-1631 ng/mL. Iron overload was present even in a non-transfusion-dependent patient and worsened in a patient post-splenectomy, suggesting a role for ineffective erythropoiesis. To further investigate this possibility, we measured erythroferrone (ERFE), GDF-15, and hepcidin levels in 2 patients so far. ERFE and GDF-15 levels were remarkably higher than controls, and hepcidin-to-ferritin ratios were significantly lower, suggesting that high ERFE, caused by ineffective erythropoiesis, inappropriately suppressed hepcidin for the body's iron levels. In summary, CDA-II diagnosis is often delayed despite early onset of anemia. Genetic testing may expedite the diagnosis. The severity of anemia is variable and there are several patients requiring transfusions. The risk of iron overload, including myocardial overload, is high and appears to be related to ineffective erythropoiesis. While frequency of transfusions may improve post-splenectomy, iron overload is not determined by blood transfusions alone and does not resolve post-splenectomy. Serum ferritin underestimated the burden of tissue iron in CDA-II, suggesting that MRI scans should be employed early. Future studies may identify a role for SEC23B deep intronic variants in CDA-II patients with heterozygous variants.

Evaluation of the main regulators of systemic iron homeostasis in pyruvate kinase deficiency

Iron homeostasis and dyserythropoiesis are poorly investigated in pyruvate kinase deficiency (PKD), the most common glycolytic defect of erythrocytes. Herein, we studied the main regulators of iron balance and erythropoiesis, as soluble transferrin receptor (sTfR), hepcidin, erythroferrone (ERFE), and erythropoietin (EPO), in a cohort of 41 PKD patients, compared with 42 affected by congenital dyserythropoietic anemia type II (CDAII) and 50 with hereditary spherocytosis (HS). PKD patients showed intermediate values of hepcidin and ERFE between CDAII and HS, and clear negative correlations between log-transformed hepcidin and log-EPO (Person’s r correlation coefficient = − 0.34), log-hepcidin and log-ERFE (r = − 0.47), and log-hepcidin and sTfR (r = − 0.44). sTfR was significantly higher in PKD; EPO levels were similar in PKD and CDAII, both higher than in HS. Finally, genotype–phenotype correlation in PKD showed that more severe patients, carrying non-missense/non-missense genotypes, had lower hepcidin and increased ERFE, EPO, and sTFR compared with the others (missense/missense and missense/non-missense), suggesting a higher rate of ineffective erythropoiesis. We herein investigated the main regulators of systemic iron homeostasis in the largest cohort of PKD patients described so far, opening new perspectives on the molecular basis and therapeutic approaches of this disease.

Modelling Congenital Dyserythropoietic Anemia Type II through Gene Editing in Hematopoietic Stem and Progenitor Cells

Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythoid lineage. They are characterized by ineffective erythropoiesis with distinct morphologic abnormalities of erythroblasts, a degree of hemolysis, and secondary hemochromatosis. Patients usually present with congenital anemia, jaundice, splenomegaly, and an absolute reticulocyte count inadequate for the degree of anemia. CDA type II (CDAII) is the most frequent type. CDAII patients show anemia of variable degrees, and 20% are transfusion dependent. The most specific finding of CDAII marrow is the presence of more than 10% mature binucleated erythroblasts with two nuclei at the same erythroid maturation stage. Treatment of CDAII patients may involve blood transfusions, iron chelation therapy and splenectomy. The only described definitive therapy is allogeneic bone marrow transplantation, which implies additional side effects for these patients. Therefore, new therapeutic approaches are needed. CDA II is caused by mutations in the SEC23B gene. SEC23B is part of coat protein complex II (COPII). COPII is involved in protein processing and Golgi-reticulum trafficking. However, how mutations in SEC23B cause CDA II is not known yet. Therefore, studying the role of SEC23B in the erythropoiesis will help to elucidate the underlying mechanism of CDA II and to develop new therapeutic approaches for the disease. We have developed a CDA II model in human cells through the introduction of genomic mutations in the gene using the CRISPR/Cas9 gene editing system. Different single guides RNAs (sgRNA) targeting the start of the coding sequence of human SEC23B gene were designed and tested in human erythroleukemia K562 cell line and in healthy human cord blood hematopoietic stem and progenitors (hCB-CD34+). The gene editing outcome at SEC23B gene was assessed at: i) genomic level through Sanger sequencing, Inference of CRISPR Edits (ICE) and Next-Generation Sequencing (NGS). ii) Protein level through Western-blot analysis. iii) Functional level through morphological analysis and erythroid differentiation either in vitro or in vivo in human hematopoietic chimeras in NOD.Cg-KitW-41JTyr+PrkdcscidIl2rgtm1Wjl/ThomJ (NBSGW) mice. K562 cells were nucleofected with three different sgRNAs, as ribonucleoprotein (RNP), independently or in combination. Afterwards, seventy five K562 clones were established from the cells nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs. Forty per cent of them showed a high efficiency of knock-out (higher than 50% of alleles). Eight SEC23BKO clones were selected for further analysis. All of those eight clones showed a reduction in SEC23B protein and six of them had a lower proliferation than control cells and morphological abnormalities, such as presence of bi/multinucleated cells. Moreover, when CB-CD34+ cells were nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs, up to 80% of knock-out efficiency and close to 90% reduction of SEC23B protein were obtained. Interestingly, when those gene edited hematopoietic progenitors were differentiated in vitro to erythroid cells, their terminal differentiation was hampered, with a reduce percentage of enucleated cells and the presence of high number of bi/multinucleated cells. Similarly, the in vivo erythroid differentiation of these gene edited progenitors two months after HSPC transplant into NBSGW mice showed again an impairment of terminal erythroid differentiation with an increment in the percentage of erythroid bi/multinucleated cells without altering other human hematopoietic lineages. In summary, CRISPR/Cas9 system has been used to model CDA II in a human cell line and in human hematopoietic progenitors through the knock-out of SEC23B gene. Our system reproduced the most relevant feature characteristic of CDA II pathology. This gene editing based CDA II model will allow the study of how mutations in SEC23B cause CDA II and the development of new therapeutic strategies to cure this disease. Disclosures Tornador: Bloodgenetics: Current Employment. Sanchez:Bloodgenetics: Current Employment. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

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

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.

Functions of the COPII gene paralogs SEC23A and SEC23B are interchangeable in vivo

Approximately one-third of the mammalian proteome is transported from the endoplasmic reticulum-to-Golgi via COPII-coated vesicles. SEC23, a core component of coat protein-complex II (COPII), is encoded by two paralogous genes in vertebrates (Sec23a and Sec23b). In humans, SEC23B deficiency results in congenital dyserythropoietic anemia type-II (CDAII), while SEC23A deficiency results in a skeletal phenotype (with normal red blood cells). These distinct clinical disorders, together with previous biochemical studies, suggest unique functions for SEC23A and SEC23B. Here we show indistinguishable intracellular protein interactomes for human SEC23A and SEC23B, complementation of yeast Sec23 by both human and murine SEC23A/B, and rescue of the lethality of sec23b deficiency in zebrafish by a sec23a-expressing transgene. We next demonstrate that a Sec23a coding sequence inserted into the murine Sec23b locus completely rescues the lethal SEC23B-deficient pancreatic phenotype. We show that SEC23B is the predominantly expressed paralog in human bone marrow, but not in the mouse, with the reciprocal pattern observed in the pancreas. Taken together, these data demonstrate an equivalent function for SEC23A/B, with evolutionary shifts in the transcription program likely accounting for the distinct phenotypes of SEC23A/B deficiency within and across species, a paradigm potentially applicable to other sets of paralogous genes. These findings also suggest that enhanced erythroid expression of the normal SEC23A gene could offer an effective therapeutic approach for CDAII patients.

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

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. DisclosuresNo relevant conflicts of interest to declare.

CD44 as a Potential Screening Marker for Preliminary Differentiation Between Congenital Dyserythropoietic Anemia Type II and Hereditary Spherocytosis.

Bone marrow examination has been the confirmatory test for congenital dyserythropoietic anemia type II (CDAII). Occasional spherocytes on peripheral blood smear can confound the diagnosis. Since a screening test is still unavailable, we explored the feasibility of using flow cytometry as a preliminary screening method. Thirteen monoclonal antibodies with specificities for eight erythrocyte membrane proteins were used in FACS analysis to probe the cellular features of red cells from CDAII, normal adults, hereditary spherocytosis (HS), and cord red cells. Confocal microscopy was performed on normal and CDAII to determine the overall distribution of CD44 and CD47. Their expression levels on cultured erythroblasts were also analyzed. The densely stained band 3 as seen in CDAII in gel electrophoresis was also obtained for Dantu phenotype. Likewise analysis of CDAII cases (n = 26) using the eosin-5'maleimide (EMA) binding test found 57% of patients giving results either positive or in the grey area for HS. Enhanced fluorescence of CD44 was detected in 96% of the CDAII patients, and anti-CD47 binding was also elevated to a lesser degree. Although RNA expressions of CD44 and CD47 in the cultured erythroblasts of normal controls and CDAII were similar, confocal microscopy revealed more CDAII red cells giving elevated fluorescence than normal red cells. A distinction between CDAII and HS can be made using the EMA Binding test and anti-CD44 binding. Confirmation of CDAII can subsequently be made based on clinical presentation together with either bone marrow examination or DNA sequencing of SEC23B. © 2016 International Clinical Cytometry Society.

Pancreatic SEC23B deficiency is sufficient to explain the perinatal lethality of germline SEC23B deficiency in mice.

In humans, loss of function mutations in SEC23B result in Congenital Dyserythropoietic Anemia type II (CDAII), a disease limited to defective erythroid development. Patients with two nonsense SEC23B mutations have not been reported, suggesting that complete SEC23B deficiency might be lethal. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration and that mice with hematopoietic SEC23B deficiency do not exhibit CDAII. We now show that SEC23B deficiency restricted to the pancreas is sufficient to explain the lethality observed in mice with global SEC23B-deficiency. Immunohistochemical stains demonstrate an acinar cell defect but normal islet cells. Mammalian genomes contain two Sec23 paralogs, Sec23A and Sec23B. The encoded proteins share ~85% amino acid sequence identity. We generate mice with pancreatic SEC23A deficiency and demonstrate that these mice survive normally, exhibiting normal pancreatic weights and histology. Taken together, these data demonstrate that SEC23B but not SEC23A is essential for murine pancreatic development. We also demonstrate that two BAC transgenes spanning Sec23b rescue the lethality of mice homozygous for a Sec23b gene trap allele, excluding a passenger gene mutation as the cause of the pancreatic lethality, and indicating that the regulatory elements critical for Sec23b pancreatic function reside within the BAC transgenes.

SEC23A Functionally Compensates for SEC23B Deficiency in Mice

Congenital Dyserythropoietic Anemia type II (CDAII) is a disease of ineffective erythropoiesis characterized by moderate anemia and increased bone marrow (BM) bi/multi-nucleated erythroid precursors. CDAII is an autosomal recessive disease resulting from mutations in SEC23B. SEC23 is a core component of COPII vesicles, which transport secretory proteins from the endoplasmic reticulum (ER) to the Golgi. Despite identification of the underlying genetic defect, the molecular mechanism by which SEC23B deficiency produces the unique CDAII phenotype remains unknown. We previously reported that mice homozygous for a Sec23b null allele die perinatally, exhibiting massive pancreatic degeneration, precluding evaluation of the adult erythroid compartment. To examine the impact of SEC23B deficiency on adult murine hematopoiesis, we generated mice with erythroid specific and pan-hematopoietic deficiency for SEC23B by crossing a second, conditional Sec23b allele (Sec23bfl), in which exons 5 and 6 are flanked by loxP sites, to an EpoR-Cre and Vav1-Cre, respectively. These mice did not exhibit anemia or any other CDAII characteristic. Similarly, mice transplanted with SEC23B-deficient fetal liver cells harvested from E17.5 embryos also failed to recapitulate any features of the CDAII phenotype. We next generated mice deficient in SEC23B exclusively in the pancreas by crossing the Sec23bfl allele to either p48-Cre or Pdx1-Cre. These mice exhibit a phenotype indistinguishable from mice with germline deletion of Sec23b, indicating that loss of pancreatic SEC23B is sufficient to explain the perinatal-lethality of global SEC23B deficiency in mice. The mammalian genome contains two paralogs for SEC23, SEC23A and SEC23B. These paralogs are highly identical at the amino acid level (~85%). We examined the relative expression of SEC23B/SEC23A in WT tissues from both humans and mice. This ratio is higher in human BM compared to pancreas, while it was higher in mouse pancreas compared to BM. In order to determine if SEC23A can rescue the phenotype of SEC23B deficient mice when expressed under the regulatory control of Sec23b, we genetically engineered the Sec23a cDNA into the endogenous genomic locus of Sec23b (Sec23a-b) in mouse embryonic stem cells via recombinase mediated cassette exchange. A heterozygous Sec23a-b intercross yielded the expected number of mice homozygous for the Sec23a-b allele. These mice exhibited normal survival, development, and fertility. Pancreas tissues dissected from Sec23a-b/a-b mice had normal weights, were histologically indistinguishable from WT controls, and did not exhibit dilated ER by transmission electron microscopy. Western blot analysis confirmed the absence of SEC23B in pancreata of Sec23a-b/a-b mice, with high levels of SEC23A expression. These data demonstrate that the SEC23A and SEC23B proteins overlap significantly (or completely) at the level of protein function, and suggest that the distinct phenotypes of human and mouse SEC23 deficiency are the result of an evolutionary shift in the tissue-specific gene expression programs of SEC23A and SEC23B. These findings also suggest that therapies that increase the expression of either SEC23 paralog in erythroid cells might be effective in CDAII. DisclosuresNo relevant conflicts of interest to declare.

SEC23B Deficiency Results in Different Phenotypes in Humans and Mice

SEC23B mutations in humans result in the autosomal recessive disease Congenital Dyserythropoietic Anemia type-II (CDAII). CDAII is characterized by moderate anemia in increased bone marrow (BM) bi/multi-nucleated erythroblasts. Despite the identification of the genetic defect underlying CDAII, the pathophysiology of this disease remains unknown. SEC23A and SEC23B are paralogous components of the coat protein complex II (COPII)-coated vesicles, which transport secretory proteins from the Endoplasmic Reticulum to the Golgi apparatus. We generated SEC23B-deficient mice and demonstrated that these animals die perinatally exhibiting massive pancreatic degeneration (Tao et. al PNAS). To examine the impact of SEC23B-deficiency on adult murine hematopoiesis, we harvested fetal liver cells (FLC), which contain hematopoietic stem cells, from SEC23B-deficient or wild-type (WT) control E17.5 embryos and transplanted them into lethally irradiated C57BL/6J mice. Recipients of SEC23B-deficient FLC did not exhibit anemia or any other CDAII characteristic (Khoriaty et. al, Mol Cell Biol), and SEC23B deficient FLC competed effectively with WT FLC at reconstituting hematopoiesis when transplanted into lethally irradiated recipient mice (Khoriaty et. al, Mol Cell Biol). A Sec23bfl conditional-allele was also generated. Mice with hematopoietic-specific SEC23B-deficiency, generated by crossing Vav1-Cre into Sec23bfl mice, also exhibited normal hematopoiesis. In contrast, mice with pancreas-specific Sec23b deficiency generated by crossing the Sec23bfl allele to a p48-Cre or Pdx1-Cre resulted in a phenotype indistinguishable from complete SEC23B-deficiency, demonstrating that loss of pancreatic Sec23b expression is sufficient to explain the perinatal lethality of SEC23B-deficient mice. To investigate different phenotypes of SEC23B deficiency in humans and mice, the SEC23B/SEC23A expression ratio was examined in murine and human tissues. This ratio is higher in mouse pancreas (12.7) compared to BM (2.6), whereas it is higher in human BM (7.8) relative to pancreas (5.5). Taken together with the high degree of amino-acid identity between SEC23A and SEC23B (~85%), these data suggest that the tissue-specific functions of SEC23A and SEC23B have shifted during evolution between humans and mice. To determine if Sec23a can rescue the lethality of SEC23B-deficient mice, we have engineered Sec23a cDNA into the endogenous genomic locus of Sec23b (Sec23A-B) via recombinase-mediated cassette exchange. A heterozygous Sec23A-B intercross is in progress. Rescue of the SEC23B deficient phenotype by SEC23A protein expressed under control of Sec23b regulatory sequences would suggest that increasing the expression of either paralog in erythroid cells might be effective in the treatment of CDAII. DisclosuresNo relevant conflicts of interest to declare.

Disparate Phenotypes Of SEC23B Deficiency In Humans and Mice

Congenital dyserythropoietic anemia type-II (CDAII) is an autosomal recessive disease characterized by moderate anemia and increased bone marrow (BM) bi/multi-nucleated erythroblasts. CDAII results from mutations in SEC23B, one of two closely related mammalian SEC23 paralogs. SEC23 is a core component of COPII coated vesicles, which transport secretory proteins from the Endoplasmic Reticulum (ER) to the Golgi apparatus. Bone marrow transplantation cures CDAII, suggesting that the pathologic defect in this disease is restricted to the hematopoietic compartment. However, the mechanism by which SEC23B-deficiency results in CDAII remains unknown. We previously reported that mice homozygous deficient for SEC23B (Sec23bgt/gt) exhibit massive pancreatic degeneration. The latter resulted in perinatal mortality precluding evaluation of the adult hematopoietic compartment. To examine the impact of SEC23B-deficiency on adult murine hematopoiesis, fetal liver cells (FLC) were harvested from Sec23bgt/gt or wildtype (WT) control E17.5 embryos and transplanted into lethally irradiated C57BL/6J mice. Recipients of Sec23bgt/gt FLC had normal peripheral blood counts and were indistinguishable from recipients of WT FLC. To test for a more subtle hematopoietic defect, Sec23bgt/gt FLCs were tested directly against WT FLC for their ability to reconstitute hematopoiesis in a competitive repopulation assay. SEC23B deficient FLC exhibited no competitive disadvantage at reconstituting erythropoiesis relative to WT FLC over 18 weeks of follow-up. Transplant of marrow from these chimeric animals into secondary recipients demonstrated continued equivalence of Sec23bgt/gt and WT hematopoietic stem cells. We also generated a second, conditional Sec23b allele, in which exons 5 and 6 are flanked by loxP sites (Sec23bfl). Deletion of exons 5 and 6 with Cre-recombinase results in a frame shift leading to a stop codon in exon 7. Mice with erythroid-specific SEC23B deficiency were generated by crossing the Sec23bfl allele to an EpoR-Cre transgene. Sec23bfl/-/EpoR-CreTg+ mice maintained normal erythropoiesis indistinguishable from their WT littermates. Pancreas-specific knock-out generated by crossing the Sec23bfl allele to p48-Cre or Pdx1-Cre transgenes generated phenotypes indistinguishable from complete SEC23B deficiency, demonstrating that loss of pancreatic Sec23b expression alone is sufficient to explain the perinatal lethality observed in Sec23bgt/gt and Sec23b-/- mice. Our results conclusively demonstrate that in contrast to humans, SEC23B-deficiency results in massive pancreatic degeneration in mice, but no CDAII in these animals.To investigate the cause of the disparate human and mouse SEC23B-deficient phenotypes, SEC23B/SEC23A expression ratios were examined in endogenous tissues from both species. This ratio (normalized to the ratio in liver mRNA as 1.0) was higher in mouse pancreas (12.7) compared to BM (2.6), with the reverse pattern observed in human BM (7.8) relative to pancreas (5.5). These data, taken together with the high degree of identity between SEC23A and SEC23B (∼ 85% amino acid identity), suggest that the tissue-specific functions of SEC23A and SEC23B may have shifted during evolution between humans and mice. To test the role of SEC23A, we generated a mouse with a conditional Sec23a allele, in which exon 3 is flanked by loxP sites (Sec23afl). Cre-recombinase mediated deletion of exon 3 results in a frame shift leading to a stop codon in exon 7 (Sec23a-). Mice with erythroid-specific SEC23A-deficiency (Sec23afl/-/EpoR-CreTg+) maintained normal red blood cell counts indistinguishable from their WT littermates.In summary, we have shown that SEC23B-deficient humans and mice exhibit disparate phenotypes. We have also demonstrated variations in the gene expression programs for SEC23A and SEC23B potentially explaining the pancreatic phenotype of SEC23B-deficiency in mice and the erythroid phenotype in humans. These results suggest that the two SEC23 paralogs have overlapping functions and that therapeutic strategies that increase the expression of either SEC23A or SEC23B in erythroid cells might be effective in CDAII. Further studies of the overlapping functions of SEC23A and SEC23B and their relevant protein cargos should provide new insight into the pathogenesis of CDAII. Disclosures:No relevant conflicts of interest to declare.

An Unusual Presentation of Congenital Dyserythropoietic Anemia Type II (CDAII) Associated with Severe Anemia in a Patient with a Novel Mutation of the SEC23B Gene

AbstractAbstract 990CDAII is an autosomal recessive disorder characterized by ineffective erythropoiesis, anemia, hemolysis, hepatosplenomegaly and morphologic abnormalities. While rare, more than 300 cases of CDAII have been described worldwide, making it the most common form of congenital dyserythropoietic anemia. In patients with CDAII, bone marrow specimens often demonstrate hypercellularity, erythroid hyperplasia and the presence of 10–45% bi- and multinucleated erythroid precursors. Generally, all patients will have a positive serum acid hemolysin test. The clinical picture includes a mild to moderate anemia and hemolysis with typical hemoglobin levels that range from 8 to 11 g/dL. In less than 10 percent of reported cases, severe anemia is seen requiring frequent red blood cell transfusions. Mutations in SEC23B are the underlying defect in the majority of patients with CDAII. SEC23B encodes an essential component of coat protein complex II coated vesicles, resulting in ineffective trafficking of secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Here we report a patient with a severe presentation of CDA II associated with a novel nonsense mutation in the SEC23Bgene.Our patient was born at 36 weeks estimated gestational age with IUGR and hypospadias. The family history included a brother born with severe anemia, severe hypospadias and hydrops fetalis who died at 4 days of life. Parents were of nonconsanguinious Samoan decent. The initial hemoglobin was 7 g/d; red blood cell transfusions were required on days of life 1, 10 and 15. Total bilirubin peaked at 3.3 and phototherapy was unnecessary. Now 20 months of age, he continues to be transfusion-dependent. Reticulocytopenia is pronounced with absolute reticulocyte counts consistently <20000/mL. Treatment with glucocorticoids produced a minimal increase in reticulocytes without a decrease in frequency of transfusions. Additional laboratory evaluation demonstrated mild indirect hyperbilirubinemia (1–3.4 mg/dL) that presented around one year of age, normal LDH and decreased haptoglobin. Bone marrow evaluation revealed dyserythropoiesis and reticulin fibrosis. Bi- and multinucleated cells were present and comprised 6% of the erythrocyte precursors.Exome sequencing was performed using Illumina hybrid capture, HiSeq sequencing and a full analysis pipeline. Sequencing revealed compound heterozygosity for two mutations in the SEC23B gene: c.53G>A (p.Arg18His) and c.1507C>T (p.Arg503X). The p.Arg18His has been previously reported in patients with CDAII. The p.Arg503X is a novel nonsense mutation that is expected to be pathogenic, likely resulting in nonsense-mediated decay of the mRNA. These mutations were confirmed clinically by Sanger sequencing and each parent was found to carry one mutation. Compound heterozygosity including a nonsense mutation in SEC23B has been reported to result in a more severe phenotype with reticulocytopenia, a finding consistent with the presentation of our patient. This case demonstrates that exome sequencing, with confirmatory dideoxy sequencing of the affected individual and the parents, can be a powerful new diagnostic approach in inherited hematologic disorders that feature genetic heterogeneity. Disclosures:No relevant conflicts of interest to declare.

Disparate SEC23B Deficient Phenotypes in Humans and Mice

Abstract 974SEC23 is a core component of the COPII coated vesicle that mediates the transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. Mutations in human SEC23B, one of two mammalian SEC23 paralogs, results in the autosomal recessive disease, congenital dyserythropoietic anemia type II (CDAII). In contrast, SEC23B deficient mice were recently reported to die perinatally with massive pancreatic degeneration, with no evidence of anemia at birth (Tao, et al., 2012. Proc.Natl.Acad.Sci.U.S.A. 109:E2001–9.) To examine the impact of SEC23B deficiency on hematopoietic function in mice surviving beyond the immediate perinatal period, lethally irradiated C57BL/6J mice were transplanted with fetal liver cells (FLC) collected from E17.5 embryos that were either wild-type (WT) or homozygous for the previously reported Sec23b gene trap allele (Sec23bgt/gt). Recipients of Sec23bgt/gt FLC were indistinguishable from their WT transplant controls and exhibited none of the phenotypic characteristics of CDAII (anemia, increased bi/multi-nucleated RBC precursors, narrower band size and increased shift of the membrane protein band 3 on SDS gel electrophoresis, or RBC double membrane appearance by electron microscopy). To test for a more subtle defect in hematopoietic reconstitution, Sec23bgt/gt FLCs were co-transplanted with WT GFP+ FLC in a 1:1 ratio, with no competitive difference observed over the course of 18 weeks of follow-up. Transplant of marrow from these chimeric animals into secondary recipients demonstrated continued equivalence of Sec23bgt/gt and WT hematopoietic stem cells. To rule out an incidental mutation in a nearby gene (“passenger gene”) as the cause of the pancreatic phenotype in SEC23B-deficient mice, two Sec23b bacterial artificial chromosome transgenes were crossed into the Sec23bgt line, with both demonstrating complete rescue of the Sec23bgt/gt pancreatic phenotype, with normal survival to adulthood. Sec23bgt/gt murine embryonal fibroblasts express a SEC23B/βGEO fusion protein consistent with the gene trap insertion into Sec23b intron 19, and this fusion protein co-immunoprecipitates with SEC24A (Tao, et al., 2012. Proc.Natl.Acad.Sci.U.S.A. 109:E2001–9). To rule out a contribution of the SEC23B/βGEO fusion protein to the disparate human and mouse SEC23B-deficient phenotypes, a second, conditional SEC23B allele was analyzed, in which exons 5 and 6 are flanked by loxP sites (Sec23bfl). Deletion of exons 5 and 6 results in frame shift and stop codon in exon 7. Mice with an erythroid-specific deficiency of SEC23B were generated by crossing the Sec23bfl allele to an EpoR-Cre transgene. Sec23bfl/-/EpoR-CreTg+ mice exhibit no anemia compared to their WT litter mates. Transplant recipients of FLCs from E16.5 embryos homozygous for a germline deletion of Sec23b exons 5 and 6 (Sec23b−/−) were also indistinguishable from mice receiving WT FLCs. Pancreas-specific knock-out generated by crossing the Sec23bfl allele to a p48-Cre or Pdx1-Cre transgene confirmed the pancreatic phenotype observed. This suggests that the perinatal lethality in Sec23b deficient mice may be the result of the loss of pancreatic Sec23b expression. To explore the mechanism for the disparate human and mouse SEC23B-deficient phenotypes, the expression patterns for SEC23A and SEC23B were examined in mice and humans by RT PCR and western blotting. The ratio of SEC23B/SEC23A expression is higher in mouse pancreas (12.7) compared to bone marrow (2.6), whereas in humans, the ratio is higher in the bone marrow (7.8) relative to pancreas (5.5) (normalized to liver). Taken together with the high degree of sequence similarity between SEC23A and SEC23B (∼ 85% identity at the amino acid level), we hypothesize that these 2 SEC23 paralogs overlap extensively at the level of protein function, with the disparate deficiency phenotypes due primarily to differences in tissue and developmentally-specific gene expression programs that have shifted extensively during recent mammalian evolution. These findings have important implications for the comparative function of other closely related paralogous genes. Further studies of the overlapping functions of SEC23A and SEC23B and their relevant protein cargos should provide new insight into the pathogenesis of CDAII and potential therapeutic approaches. Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

In Vitro Characterization of R14W Mutation in SEC23B, the CDAII Causative Gene

Abstract 2098Congenital dyserythropoietic anemias (CDAs) designate a group of genetic disorders in which ineffective erythropoiesis is the predominant mechanism of anemia marked by distinct morphologic abnormalities of the majority of erythroblasts in the bone marrow. CDA type II (CDAII) is the most common type of CDA. It is characterized by a recessive model of inheritance, mild to moderate anemia, jaundice, and splenomegaly (Fukuda MN, Glycobiology 1990; Fukuda MN, Clin Haematol 1993), by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance (Schwarz K and Iolascon A. et al., Nat Genet. 2009). The specific hallmark of diagnosis is the presence of the more abundant protein of membrane red cell, band 3, in a hypoglycosilated state; this is thinner and migrated slightly faster than in controls on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Anselstetter V et al., Br J Haematol 1977).The causative gene of CDAII, SEC23B, is a member of the SEC23 subfamily of the SEC23/SEC24 family, which is involved in vesicle trafficking. The encoded protein has similarity to yeast Sec23p (66.4%) component of COPII, the coat protein complex responsible for vesicle budding from the ER. The function of this gene product has been implicated in cargo selection and concentration.The SEC23B gene spans approximately 54 kb on human chromosome 20p11.23 and codifies for a protein of 767 aminoacids divided in five functional domains: zink finger, trunk, β-sheet, helical and gelsolin domain. Although most of the SEC23B mutations are sporadic events, 4 mutations (R14W, E109K, R497C, I318T) accounted for more than 50% of mutated alleles. The aim of this study is the in vitro characterization of the R14W mutation, the most frequent variant in Italy, particularly in South of Italy (Russo R et al., Am J Hematol. in press). We used the human erythroleukemia cell line, HEL, as in vitro model because it is more similar to mature red cell. By using in silico tool ESyPred3D Web Server 1.0, we predicted that this aminoacidic substitution alters the zink finger domain 3D structure, when compared to wild type protein. This tool implements homology modeling approach followed by a final analysis with MODELLER release 4 in order to build a 3D model of the submitted protein (Lambert et al, 2002). However, when we transfected the SEC23B-R14W we observed a strong reduction of gene expression in the mutant when compared to SEC23B-wt construct by qRT-PCR. These results have been also confirmed at the protein level. In fact the protein expression of SEC23B-R14W showed a reduction comparable to gene expression respect to SEC23B-wt construct. Immunofluorescence analyses by confocal microscopy, were used for the investigation of the cellular localization of SEC23B-R1W protein and, interestingly, the localization of mutant protein was not changed when compared to that wt. Our data allow us to hypothesize that the mutation R14W gives rise to anomalous protein product quantity, but the protein function would be like not altered.Our findings demonstrated that the most frequent mutation found in Italy, SEC23B-R14W, results in a reduced half-life of the mutated mRNA, without altering the cellular localization in HEL cell line. SEC23B belongs to a multiproteic compelx that assembles with the others complex proteins in accordance with a specific structure. Each structure establishes a cargo selectivity. Further studies are necessary in order to understand what is the role of SEC23B in selectivity of the cargo in erythroid cells and how its disruption could determines the appearance of the principal pathological phenotype in CDAII patients, for example the hypoglycosilation of band 3. Disclosures:No relevant conflicts of interest to declare.

Molecular Analysis of the SEC23B Gene In Patients Affected by Congenital Dyserythropoietic Anemia Type II (CDAII)

AbstractAbstract 4227CDAII, the most frequent type of congenital dyserythropoietic anemia, is an autosomal recessive disease characterized by ineffective erythropoiesis, peripheral hemolysis, erythroblast morphological abnormalities and hypoglycosylation of some RBC membrane proteins. Last year we and others identified SEC23B as the gene responsible for CDAII (Schwarz et al, 2009, Bianchi et al, 2009). SEC23B is a member of the SEC23/SEC24 family, a component of COPII coat protein complex which is involved in protein trafficking through membrane vesicles from the endoplasmic reticulum to the Golgi apparatus. The gene, localized on chromosome 20p11, is split in 20 exons and codifies for a 767 aa protein. The aim of the study was to characterize the molecular defect in a large series of CDAII patients of Caucasian origin. Fifty-one CDAII patients from 49 unrelated families (17 Italians, 20 German or Swiss, 4 Dutch, 2 British, 2 Czech and 1 Greek, Turkish, Bulgarian and Irish origin) were analyzed by direct exon sequencing. We identified 36 different mutations, 25 of which were not described before. Eighteen were disruptive and 18 were missense mutations. The patients' molecular data are summarized in the Figure (novel mutations are reported in bold). All the missense mutations affected highly conserved aminoacids and were not found in 200 normal alleles examined. The c.325G>A mutation was identified in 15 homozygous patients and in two cases in combination with other mutations. The change c.40C>T was detected in 16 unrelated patients as heterozygous mutation and, for the first time, at the homozygous level in one patient only. Considering the entire series of patients (including previously published cases) characterized by our groups (86 CDAII patients from 77 unrelated families) c.325G>A and c.40C>T mutations account for 49% (76/154) of the unrelated mutated alleles and therefore should be firstly screened during molecular diagnosis of CDAII. A c.325G>A mutation usually results in a mild to moderate clinical picture at homozygous level, whereas it may cause a very severe clinical pictures when combined with other mutations (Fermo, et al 2010). Despite the sequencing of all exons and flanking intronic regions, 5 patients displayed only one mutation, suggesting the possibility that mutations could be located in regulatory regions or that a second gene could be involved in the pathogenesis of CDAII.P. Bianchi and K. Schwarz contributed equally to this work. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Mutations affecting the secretory COPII coat component SEC23B cause congenital dyserythropoietic anemia type II

Congenital dyserythropoietic anemias (CDAs) are phenotypically and genotypically heterogeneous diseases. CDA type II (CDAII) is the most frequent CDA. It is characterized by ineffective erythropoiesis and by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance. Other features of the peripheral red blood cells are protein and lipid dysglycosylation and endoplasmic reticulum double-membrane remnants. Development of other hematopoietic lineages is normal. Individuals with CDAII show progressive splenomegaly, gallstones and iron overload potentially with liver cirrhosis or cardiac failure. Here we show that the gene encoding the secretory COPII component SEC23B is mutated in CDAII. Short hairpin RNA (shRNA)-mediated suppression of SEC23B expression recapitulates the cytokinesis defect. Knockdown of zebrafish sec23b also leads to aberrant erythrocyte development. Our results provide in vivo evidence for SEC23B selectivity in erythroid differentiation and show that SEC23A and SEC23B, although highly related paralogous secretory COPII components, are nonredundant in erythrocyte maturation.

Congenital dyserythropoietic anemia type II (CDAII) is caused by mutations in theSEC23Bgene

Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease characterized by ineffective erythropoiesis, hemolysis, erythroblast morphological abnormalities, and hypoglycosylation of some red blood cell (RBC) membrane proteins. Recent studies indicated that CDAII is caused by a defect disturbing Golgi processing in erythroblasts. A linkage analysis located a candidate region on chromosome 20, termed the CDAN2 locus, in the majority of CDAII patients but the aberrant gene has not so far been elucidated. We used a proteomic-genomic approach to identify SEC23B as the candidate gene for CDAII by matching the recently published data on the cytoplasmic proteome of human RBCs with the chromosomic localization of CDAN2 locus. Sequencing analysis of SEC23B gene in 13 CDAII patients from 10 families revealed 12 different mutations: six missense (c.40C>T, c.325G>A, c.1043A>C, c.1489C>T, c.1808C>T, and c.2101C>T), two frameshift (c.428_428delAinsCG and c.1821delT), one splicing (c.689+1G>A), and three nonsense (c.568C>T, c.649C>T, and c.1660C>T). Mutations c.40C>T and c.325G>A were detected in unrelated patients. SEC23B is a member of the Sec23/Sec24 family, a component of the COPII coat protein complex involved in protein transport through membrane vesicles. Abnormalities in this gene are likely to disturb endoplasmic reticulum (ER)-to-Golgi trafficking, affecting different glycosylation pathways and ultimately accounting for the cellular phenotype observed in CDAII.

Abnormalities of erythrocyte glycoconjugates are identical in two families with congenital dyserythropoietic anemia type II with different chromosomal localizations of the disease gene

We analyzed erythrocyte glycoconjugates in two families with congenital dyserythropoietic anemia type II (CDA-II): family 2 with the typical localization of the disease gene to chromosome 20q11.2 and family 1 in which this localization was excluded. Despite the different genetics, the erythrocyte glycoconjugate abnormalities in the two families were identical suggesting a complex inheritance of CDA-II. We also found that erythrocyte anion exchanger 1 protein is decreased in CDA-II homozygotes and obligate carriers alike.