Congenital Dyserythropoietic Anemia Type Research Articles

Unveiling the genetic landscape of suspected congenital dyserythropoietic anemia type I: A retrospective cohort study of 36 patients.

Congenital Dyserythropoietic Anemia type I (CDA I) is a rare hereditary condition characterized by macrocytic/normocytic anemia, splenomegaly, iron overload, and distinct abnormalities during late erythropoiesis, particularly internuclear bridges between erythroblasts. Diagnosis of CDA I remains challenging due to its rarity, clinical heterogeneity, and overlapping phenotype with other rare hereditary anemias. In this case series, we present 36 patients with suspected CDA I. A molecular diagnosis was successfully established in 89% of cases, identifying 16 patients with CDA I through the presence of 18 causative variants in the CDAN1 or CDIN1 genes. Transcriptomic analysis of CDIN1 variants revealed impaired erythroid differentiation and disruptions in transcription, cell proliferation, and histone regulation. Conversely, 16 individuals received a different diagnosis, primarily pyruvate kinase deficiency. Comparisons between CDA I and non-CDA I patients revealed no significant differences in erythroblast morphological features. However, hemoglobin levels and red blood cell count differed between the two groups, with non-CDA I subjects being more severely affected. Notably, most patients with severe anemia belonged to the non-CDA I group (82% non-CDA I vs. 18% CDA I), with a subsequent absolute prevalence of transfusion dependency among non-CDA I patients (100% vs. 41.7%). All patients exhibited reduced bone marrow responsiveness to anemia, with a more pronounced effect observed in non-CDA I patients. Erythropoietin levels were significantly higher in non-CDA I patients compared to CDA I patients. However, evaluations of erythroferrone, soluble transferrin receptor, and hepcidin revealed no significant differences in plasma concentration between the two groups.

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.

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.

Safety and Efficacy of Mitapivat in Adult Patients with Erythrocyte Membranopathies (SATISFY)

Background: Membranopathies encompass hemolytic disorders arising from genetic defects in erythrocyte membrane proteins and include hereditary spherocytosis and stomatocytosis. Congenital dyserythropoietic anemia type II (CDA II) is associated with SEC23B gene variants and its phenotype may be similar to hereditary spherocytosis. Anemia with or without the need for chronic transfusion, jaundice, splenomegaly, iron overload are complications common in both hereditary membranopathies and CDA II. Current treatment options for membranopathies and CDA II 1, apart from splenectomy, are primarily supportive. Mitapivat, an investigational pyruvate kinase-R (PKR) activator, has demonstrated efficacy in improving anemia and reducing hemolysis in adult patients with PK deficiency, thalassemia, sickle cell disease, and a mouse model of hereditary spherocytosis. Study Design and Methods: This study (NCT05935202) is an investigator initiated, prospective, multicenter, single-arm phase 2 trial. The study will be conducted in the European Union in Denmark and The Netherlands. It is the first clinical study in the context of the European Reference Network EuroBloodNet and will be sponsored by its closely associated non-for-profit EuroBloodNet Association. A sibling study in Toronto, Canada will be registered separately, and data will be pooled in a prespecified statistical analysis plan. The study will include approximately twenty-five adult patients (aged 18 years and older) diagnosed with a membranopathy or CDA II. Diagnosis must be confirmed genetically. Average hemoglobin concentration (Hb) must be less than 13.0 g/dL for males and 11.0 g/dL for females. Patients with average Hb >10.0 g/dL for males and females at screening must meet at least one of the following additional criteria: Splenomegaly, fatigue attributed to hemolysis, or paraclinical hemolysis. Adequate organ function is required. Patients cannot be included if they have a diagnosis of PK deficiency or if they have received red blood cell (RBC) transfusions (≥5 units the last 12 months or any within the last 3 months). During the 8-week Dose Escalation Period, subjects will receive an initial dose of 50 mg mitapivat twice daily (BID). Based on safety and changes in Hb levels, dosing may be increased to 100 mg BID at week 4. Patients who tolerate mitapivat may be eligible to continue in two consecutive 24-week Fixed Dose Periods, with an interim analysis in between the two fixed dose periods. The primary objective of this study is to evaluate the safety of mitapivat, assessed through the occurrence of treatment-emergent adverse events. Secondary objectives include assessing the effects of mitapivat on Hb concentration (≥1.0 g/dL increase sustained at two scheduled visits), hemolysis, erythropoiesis, patient-reported outcome measures, and spleen size. Exploratory endpoints include enzyme activity and stability of PK, Hb oxygen affinity, proteomics and metabolomics. To assess how activation of PK will impact RBC function we will measure RBC deformability (osmotic gradient ektacytometry and cell membrane stability) in whole blood and in different RBC subpopulations. Acknowledgement: This project is carried out within the framework of European Reference Network on Rare Haematological Diseases (ERN-EuroBloodNet)-Project ID No 101085717. ERN-EuroBloodNet is partly co-funded by the European Union within the framework of the Fourth EU Health Programme. The study is funded by a grant from Agios Pharmaceuticals.

A stepwise diagnostic approach for undiagnosed Anemia in children: A model for low-middle income country

BackgroundReaching a precise diagnosis in rare inherited anemia is extremely difficult and challenging, especially in areas with limited use of genetic studies, which makes undiagnosed anemia a unique clinical entity in tertiary hematology centers. In this study, we aim at plotting a stepwise diagnostic approach in children with undiagnosed anemia while identifying indications for genetic testing. Patients and methodsA one-year cross-sectional study involved 44 children and adolescents with undiagnosed anemia after undergoing an initial routine panel of investigations. They were classified based on mean corpuscular volume (MCV) into 3 groups: microcytic (n = 19), normocytic (n = 14) and macrocytic (n = 11). An algorithm that included four levels of investigations was devised for each category. ResultsAfter applying a systematic diagnostic approach, 33 patients (75 %) were diagnosed of whom 7 (15 %) had combined diagnoses, while 11 (25 %) patients remained undiagnosed. Based on the first, second, third and fourth levels of investigations, patients were diagnosed, respectively, as follows: of the 11 patients, 7 were microcytic, 3 normocytic and 1 macrocytic; of the 7 patients, 2 were microcytic, 2 normocytic, and 3 macrocytic; of 10 patients, 5 were microcytic, 4 normocytic and 1 macrocytic; finally, of the 16 patients, 8 were microcytic, 6 normocytic and 2 macrocytic. Numbers recorded appear higher than the actual number of the patients because some of them were diagnosed by more than one level of investigation. The diagnoses obtained in the microcytic group showed hemoglobinopathies, iron refractory iron deficiency anemia (IRIDA), membrane defects, sideroblastic anemia, hypo-transferrinemia, a combined diagnosis of sickle cell trait and pyropoikilocytosis. The diagnoses also showed a combined diagnosis of hereditary spherocytosis (HS) and alpha thalassemia minor, and a combined diagnosis of iron deficiency anemia and beta thalassemia minor, while 15 % remained undiagnosed. In the normocytic group, the diagnosis revealed autosomal recessive (AR) HS, vitamin B12 deficiency, pyruvate kinase deficiency (PKD), congenital dyserythropoietic anemia (CDA) type I, Diamond Blackfan anemia and beta thalassemia major. In addition, it showed a combined diagnosis of AR HS and CDA type II, a combined diagnosis of AR HS and PKD, and a combined diagnosis of dehydrated stomatocytosis (DHS) and G6PD carrier, meanwhile 20 % remained undiagnosed. Finally, the macrocytic group was diagnosed by vitamin B12 deficiency, sideroblastic anemia, PKD, a combined diagnosis of PKD and G6PD deficiency carrier, while 45 % remained undiagnosed. ConclusionConducting a stepwise approach with different levels of investigations may help reach the diagnosis of difficult anemia without having to resort to unnecessary investigations. Combined diagnosis is an important cause of undiagnosed anemia, especially in countries with high frequency of consanguinity. The remaining 25 % of the patients continued to be undiagnosed, requiring more sophisticated investigations.

Development of High-Resolution Melting Curve Analysis for rapid detection of SEC23B gene mutation causing Congenital Dyserythropoietic Anemia type II in Indian population

BackgroundCongenital dyserythropoietic anemias (CDAs) are a very rare and heterogeneous group of disorders characterized by ineffective erythropoiesis. CDA II is caused by mutations in the SEC23B gene. The most common mutation reported in India is c.1385 A > G, p.Y462C. There is no simple and cost-effective confirmatory diagnostic test available for CDA, and therefore, many patients remain undiagnosed. High-resolution melting curve (HRM) analysis is a polymerase chain reaction (PCR) based technique applied to identify genetic differences and scan nucleic acid sequences. HRM can be used to rapidly screen the common mutation causing CDA II in the Indian population. Thus, we studied the use of High-Resolution Melting Curve Analysis to detect common mutation causing CDA II in the Indian population.Method11 patients having SEC23B (Y462C) mutation causing CDA II are considered for this study. HRM was used to check the presence of Y462C mutation. To verify the accuracy of the HRM analysis, we compared HRM results with the results of Sanger sequencing. This helped us to confirm the diagnosis.ResultsWe have described the clinical, hematological, and genetic data of eleven patients suffering from CDAII. According to HRM and Sanger sequencing, a homozygous SEC23B (Y462C) mutation was present in all patients, whereas a heterozygous Y462C mutation was present in their parents.ConclusionOur data showed that High-Resolution Melting (HRM) analysis could be used to rapidly screen common SEC23B mutation that causes CDA II in the Indian population.

Modelling the erythroblastic island niche of dyserythropoietic anaemia type IV patients using induced pluripotent stem cells.

Introduction: Congenital dyserythropoietic anaemia (CDA) type IV has been associated with an amino acid substitution, Glu325Lys (E325K), in the transcription factor KLF1. These patients present with a range of symptoms, including the persistence of nucleated red blood cells (RBCs) in the peripheral blood which reflects the known role for KLF1 within the erythroid cell lineage. The final stages of RBCs maturation and enucleation take place within the erythroblastic island (EBI) niche in close association with EBI macrophages. It is not known whether the detrimental effects of the E325K mutation in KLF1 are restricted to the erythroid lineage or whether deficiencies in macrophages associated with their niche also contribute to the disease pathology. Methods: To address this question, we generated an in vitro model of the human EBI niche using induced pluripotent stem cells (iPSCs) derived from one CDA type IV patient as well as two iPSC lines genetically modified to express an KLF1-E325K-ERT2 protein that could be activated with 4OH-tamoxifen. The one patient iPSC line was compared to control lines from two healthy donors and the KLF1-E325K-ERT2 iPSC line to one inducible KLF1-ERT2 line generated from the same parental iPSCS. Results: The CDA patient-derived iPSCs and iPSCs expressing the activated KLF1-E325K-ERT2 protein showed significant deficiencies in the production of erythroid cells with associated disruption of some known KLF1 target genes. Macrophages could be generated from all iPSC lines but when the E325K-ERT2 fusion protein was activated, we noted the generation of a slightly less mature macrophage population marked by CD93. A subtle trend in their reduced ability to support RBC enucleation was also associated with macrophages carrying the E325K-ERT2 transgene. Discussion: Taken together these data support the notion that the clinically significant effects of the KLF1-E325K mutation are primarily associated with deficiencies in the erythroid lineage but it is possible that deficiencies in the niche might have the potential to exacerbate the condition. The strategy we describe provides a powerful approach to assess the effects of other mutations in KLF1 as well as other factors associated with the EBI niche.

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.

The role of perinatal phenotyping in confirming the molecular diagnosis of congenital dyseryhtropoietic anaemia type I (CDA1) – a case report

Congenital dyserythropoietic anaemia type I (CDA1) is an autosomal recessive disorder characterised by macrocytic anaemia and ineffective erythropoiesis. A family with a prenatal presentation of severe hydrops fetalis at 20 weeks gestation were referred for trio whole exome sequencing, which identified a homozygous variant in the CDAN1 gene (NM_138477.2:c.3107C>T, p.(Ser1036Phe)), which was classified as a variant of uncertain significance based on the following ACMG criteria: PS4_supporting, PM2_supporting and PP3.

Ultrastructural characteristics of erythroid cells in congenital dyserythropoietic anemia type II, with a focus on peripheral cisternae and double membranes.

Peripheral cisternae and double membranes (PCDMs) in erythroid cells are a landmark of type II congenital dyserythropoietic anemia (CDA). To gain further insights into the mechanism of dyserythropoiesis, erythroblasts and erythrocytes in bone marrow were studied in 22 Chinese patients with CDA Ⅱ by transmission electron microscopy. The study demonstrated an increase in all patients in erythroblasts with PCDMs with development from pro-erythroblast to red blood cells. PCDMs often connected with cisternae of endoplasmic reticulum (ER) and the perinuclear space, and were accompanied by karyopyknosis, karyolysis and disruption in polychromatic and orthochromatic erythroblasts. The results suggest that PCDMs are transformed from ER during erythropoiesis and participate in the dissolution and deletion of late erythroid cells in patients with CDA II.

Hematopoietic Cell Transplantation for Congenital Dyserythropoietic Anemia: A Report from the Pediatric Transplant and Cellular Therapy Consortium

Hematopoietic cell transplantation (HCT) is the sole curative option for congenital dyserythropoietic anemia (CDA), a rare type of hemolytic anemia characterized by anemia, ineffective erythropoiesis, and secondary hemochromatosis. In this retrospective multicenter study, wereport the outcomes of children with CDA who underwent HCT at participating Pediatric Transplantation and Cellular Therapy Consortium centers. Clinical information on HCT and associated outcomes was collected retrospectively using a common questionnaire. Data were analyzed using descriptive statistics and appropriate analysis. Eighteen patients with CDA who underwent allogeneic HCT between 2002 and 2020 were identified. The majority of patients (n= 13) had CDA type II, and the remainder had either CDA type I (n=2) or CDA of unknown type (n=3). Mutations were identified in 7 patients (39%), includingSEC23Bin 5,GATA1in 1, and abnormality of chromosome 20 in 1. Thirteen patients had evidence of iron overload pre-HCT and received chelation therapy for a median duration of 10 months (range, 2 months to 17 years) pre-HCT. The median age at the time of HCT was 5.5 years (range, 0.7 to 26 years). Donors were HLA-matched (sibling, 4; unrelated, 10) and mismatched (haploidentical, 1; unrelated, 3). Graft sources were bone marrow in 15 patients, umbilical cord blood in 2 patients, or both in 1 patient. Conditioning included busulfan-based myeloablative (67%), fludarabine-based reduced-intensity (27%), or nonmyeloablative (6%) regimens. Five patients developed veno-occlusive disease, and 4 had viral reactivation. The cumulative incidence of acute graft-versus-host disease (GVHD) was 33%, and that of chronic GVHD was 22%. Four patients (22%) experienced graft failure; all engrafted following either a second HCT (n=2) or third HCT (n=2) but sustained considerable morbidities (3 GVHD, 1 death, 2 viral reactivation). With a median follow-up of 3.2 years (range, 0.6 to 14 years)), the 2-year overall survival, event-free survival (EFS), and GVHD-free EFS were 88% (95% confidence interval [CI], 73% to 100%), 65% (95% CI, 45% to 92%), and 60% (95% CI, 40% to 88%), respectively.Univariate analysis did not identify any patient- or transplantation-related variables impacting outcomes. Our study indicates that HCT can be curative for patients with CDA. Strategies such as aggressive chelation, use of preconditioning therapy, and early HCT in the presence of a suitable donor before comorbidities occur are needed to improve engraftment without increasing the risk for toxicity and mortality.

Multiple oral and cerebral relapses of a Granular cell tumor (Abrikossoff Tumor) in a young girl affected by congenital dyserythropoietic anemia type II.

A 14-year-old girl presented with 1 cm large whitened lesion on the ventral surface of the tongue, appeared from 1 month. Past history showed congenital dyserythropoietic anemia type II. The lesion was excised and microscopic and immunohistochemical analyses were compatible with benign Abrikossoff tumor. Total body MRI was negative. After six months the patient presented a second tongue lesion and four months later another large painful lesion in the soft palate, with the same istological diagnosis. In addition, she had other multiple lesions: two apperead at pharyngeal level (not biopsied) that remain stable over time, and one at the pituitary gland. Granular cell tumors, with or without multiple lesions, are rare in children. About 50% of cases involve the head and neck region, with the tongue being the most affected site. Therapy is based on the surgical excision of the lesions; however some tumor forms, although their histological aspect of benignity, often have an important infiltrative power, making the therapeutic approach difficult, as in our case.

SEC23B Loss-of-Function Suppresses Hepcidin Expression by Impairing Glycosylation Pathway in Human Hepatic Cells.

Biallelic pathogenic variants in the SEC23B gene cause congenital dyserythropoietic anemia type II (CDA II), a rare hereditary disorder hallmarked by ineffective erythropoiesis, hemolysis, erythroblast morphological abnormalities, and hypo-glycosylation of some red blood cell membrane proteins. Abnormalities in SEC23B, which encodes the homonymous cytoplasmic COPII (coat protein complex II) component, disturb the endoplasmic reticulum to Golgi trafficking and affect different glycosylation pathways. The most harmful complication of CDA II is the severe iron overload. Within our case series (28 CDA II patients), approximately 36% of them exhibit severe iron overload despite mild degree of anemia and slightly increased levels of ERFE (the only erythroid regulator of hepcidin suppression). Thus, we hypothesized a direct role of SEC23B loss-of-function in the pathomechanism of hepatic iron overload. We established a hepatic cell line, HuH7, stably silenced for SEC23B. In silenced cells, we observed significant alterations of the iron status, due to both the alteration in BMP/SMADs pathway effectors and a reduced capability to sense BMP6 stimulus. We demonstrated that the loss-of-function of SEC23B is responsible of the impairment in glycosylation of the membrane proteins involved in the activation of the BMP/SMADs pathway with subsequent hepcidin suppression. Most of these data were confirmed in another hepatic cell line, HepG2, stably silenced for SEC23B. Our findings suggested that the pathogenic mechanism of iron overload in CDA II is associated to both ineffective erythropoiesis and to a specific involvement of SEC23B pathogenic variants at hepatic level. Finally, we demonstrated the ability of SEC23B paralog, i.e., SEC23A, to rescue the hepcidin suppression, highlighting the functional overlap between the two SEC23 paralogs in human hepatic cells.

3131 – KLF1-E325K MUTANT PROTEIN FOUND IN PATIENTS WITH CONGENITAL DYSERYTHROPOIETIC ANEMIA RESULTS IN GENETIC DISARRAY IN ERYTHROBLASTIC ISLAND MACROPHAGES, WITH NO OBSERVED EFFECT ON FUNCTION

While the E325K mutation in the transcription factor KLF1 is known to cause morphological defects and gene dysregulation in red blood cells (RBCs), resulting in the disease congenital dyserythropoietic anemia (CDA) type IV, the effect of the mutation on other cell types has not yet been investigated. RBCs develop in erythroblastic islands (EBIs), structures found within the bone marrow consisting of a central macrophage cell surrounded by developing erythroblasts. The macrophage compartment of the EBI niche has been implicated in several RBC disorders, but it was not known whether it is affected by the KLF1-E325K mutation. This question was addressed in two ways. Firstly, to investigate the effect of the mutation on the expression of KLF1 target genes, bulk RNA-sequencing was performed on wild-type iPSC-derived macrophages and mutant iPSC-macrophages from both a CDA patient-derived iPSC line and a KLF1-E325K inducible iPSC line. Secondly, to investigate any functional effects on RBC enucleation and maturation these iPSC-derived macrophages were used in an in vitro model of the human EBI niche. WT-KLF1 and E325K-KLF1 are known to have differences in their ability to activate target genes in RBCs, and this was also observed here in macrophages. For example, FDCSP, encoding a secreted peptide, is upregulated by WT-KLF1 but not by KLF1-E325K. However, using our model of the EBI, E325K mutant macrophages had no detectable functional defects and supported the maturation and enucleation of RBCs at comparable levels to wild-type macrophages. While the E325K mutation in the transcription factor KLF1 is known to cause morphological defects and gene dysregulation in red blood cells (RBCs), resulting in the disease congenital dyserythropoietic anemia (CDA) type IV, the effect of the mutation on other cell types has not yet been investigated. RBCs develop in erythroblastic islands (EBIs), structures found within the bone marrow consisting of a central macrophage cell surrounded by developing erythroblasts. The macrophage compartment of the EBI niche has been implicated in several RBC disorders, but it was not known whether it is affected by the KLF1-E325K mutation. This question was addressed in two ways. Firstly, to investigate the effect of the mutation on the expression of KLF1 target genes, bulk RNA-sequencing was performed on wild-type iPSC-derived macrophages and mutant iPSC-macrophages from both a CDA patient-derived iPSC line and a KLF1-E325K inducible iPSC line. Secondly, to investigate any functional effects on RBC enucleation and maturation these iPSC-derived macrophages were used in an in vitro model of the human EBI niche. WT-KLF1 and E325K-KLF1 are known to have differences in their ability to activate target genes in RBCs, and this was also observed here in macrophages. For example, FDCSP, encoding a secreted peptide, is upregulated by WT-KLF1 but not by KLF1-E325K. However, using our model of the EBI, E325K mutant macrophages had no detectable functional defects and supported the maturation and enucleation of RBCs at comparable levels to wild-type macrophages.

Congenital dyserythropoietic anemia type IV with kruppel-like factor 1 E325K mutation in a preterm neonate: Case and literature review

Congenital dyserythropoietic anemia type IV with kruppel-like factor 1 E325K mutation in a preterm neonate: Case and literature review

A common human missense mutation of vesicle coat protein SEC23B leads to growth restriction and chronic pancreatitis in mice

SEC23B is one of two vertebrate paralogs of SEC23, a key component of the coat protein complex II vesicles. Complete deficiency of SEC23B in mice leads to perinatal death caused by massive degeneration of professional secretory tissues. However, functions of SEC23B in postnatal mice and outside professional secretory tissues are unclear. In this study, we generated a Sec23b KO mouse and a knockin (KI) mouse with the E109K mutation, the most common human mutation in congenital dyserythropoietic anemia type II patients. We found that E109K mutation led to decreases in SEC23B levels and protein mislocalization. However, Sec23bki/ki mice showed no obvious abnormalities. Sec23b hemizygosity (Sec23bki/ko) was partially lethal, with only half of expected hemizygous mice surviving past weaning. Surviving Sec23bki/ko mice exhibited exocrine insufficiency, increased endoplasmic reticulum stress and apoptosis in the pancreas, and phenotypes consistent with chronic pancreatitis. Sec23bki/ko mice had mild to moderate anemia without other typical congenital dyserythropoietic anemia type II features, likely resulting from exocrine insufficiency. Moreover, Sec23bki/ko mice exhibited severe growth restriction accompanied by growth hormone (GH) insensitivity, reminiscent of Laron syndrome. Growth restriction is not associated with hepatocyte-specific Sec23b deletion, suggesting a nonliver origin of this phenotype. We propose that inflammation associated with chronic pancreatic deficiency may explain GH insensitivity in Sec23bki/ko mice. Our results reveal a genotype–phenotype correlation in SEC23B deficiency and indicate that pancreatic acinar is most sensitive to SEC23B deficiency in adult mice. The Sec23bki/ko mice provide a novel model of chronic pancreatitis and growth retardation with GH insensitivity.

The congenital dyserythropoieitic anemias: genetics and pathophysiology.

The congenital dyserythropoietic anemias (CDA) are hereditary disorders characterized by ineffective erythropoiesis. This review evaluates newly developed CDA disease models, the latest advances in understanding the pathogenesis of the CDAs, and recently identified CDA genes. Mice exhibiting features of CDAI were recently generated, demonstrating that Codanin-1 (encoded by Cdan1) is essential for primitive erythropoiesis. Additionally, Codanin-1 was found to physically interact with CDIN1, suggesting that mutations in CDAN1 and CDIN1 result in CDAI via a common mechanism. Recent advances in CDAII (which results from SEC23B mutations) have also been made. SEC23B was found to functionally overlap with its paralogous protein, SEC23A, likely explaining the absence of CDAII in SEC23B-deficient mice. In contrast, mice with erythroid-specific deletion of 3 or 4 of the Sec23 alleles exhibited features of CDAII. Increased SEC23A expression rescued the CDAII erythroid defect, suggesting a novel therapeutic strategy for the disease. Additional recent advances included the identification of new CDA genes, RACGAP1 and VPS4A, in CDAIII and a syndromic CDA type, respectively. Establishing cellular and animal models of CDA is expected to result in improved understanding of the pathogenesis of these disorders, which may ultimately lead to the development of new therapies.

Whole exome sequencing analysis of compound heterozygous variants of CDAN1 gene in a Chinese family with non-immune hydrops fetalis

To study the clinical characteristics and genetic variants in a family with non-immune hydrops fetalis. Peripheral blood samples were collected from a pregnant woman with suspected non-immune hydrops fetalis of the fetus for routine blood analysis, Rh typing and TORCH test. Amniotic fluid sample was collected for G-banded chromosomal karyotyping. The genomic DNA of the proband was extracted for analysis of chromosomal abnormalities using copy number variation sequencing. Whole-exome sequencing (Trios-WES) was performed on Illumina NovaSeq 6000 platform and exonic DNA was enriched using Agilent Sure Select XT Human All Exon V6. Sorting intolerant from tolerant (SIFT), I-mutant2, PolyPhen-2 and PROVEAN were used to predict the potential effects of amino acid substitution on protein function and splicing variation. The spatial structure of codanin-1 was modeled and visualized with Alpha Fold 2 and PyMOL 2.3 software, and the variants with potential clinical significance were confirmed by Sanger sequencing. Fetal ultrasound at 17 weeks of gestation showed extensive subcutaneous edema, ascites, pleural effusion, enlarged liver and spleen, thickened placenta and pericardium defect. NGS reveals that proband has carried c.2140C>T, p.R714W, and c.1264_1265delCT, p.L422* compound heterozygous variants of CDAN1 gene, which were found to be pathogenic and inherited from proband's father and mother respectively. We identified a novel heterozygous CDAN1 gene mutation causing fetal-onset congenital dyserythropoietic anemia type 1, which triggers non-immune hydrops fetalis.

EKLF/Klf1 Regulates Erythroid Transcription By Its Pioneering Activity and Subsequent Control of RNA Pol II Pause-Release

EKLF/Klf1 is a master transcriptional activator of critical genes that regulate both erythroid fate specification and terminal erythroid maturation. EKLF binds to DNA using three Zn-fingers at its C-terminus while the N-terminus constitutes a transcription activation domain (TAD) that interacts with various transcription co-factors including the protein acetylase CBP. An autosomal semi-dominant mutation at a single residue (E339D) in the mouse EKLF Zn-finger leads to Neonatal anemia (Nan). A mutation at the same residue in human EKLF (E325K) causes Congenital Dyserythropoietic Anemia type IV (CDA IV). Nan/Nan mice show lethality at embryonic day E10-11, in contrast to EKLF-/- homozygotes that survive until E15. Nan/+ heterozygotes survive to adulthood but are severely anemic, unlike EKLF+/- heterozygotes that display no aberrant phenotypes. The Nan-EKLF protein has an altered DNA binding specificity leading to a vastly altered transcriptome by two mechanisms. First, Nan-EKLF binding causes ectopic gene expression that significantly contributes to the severe anemia in Nan/+. Second, a subset of EKLF targets is downregulated in heterozygous Nan/+ mutants despite the presence of one copy of wild type EKLF, exacerbating the anemia. Thus, uncovering the mechanism by which gene expression is altered in Nan/+ may illuminate how EKLF normally activates transcription of its targets in vivo.To this end, we first examined the global occupancy of RNA Pol II phospho-Ser5 (as a paused mark) and phospho-Ser2 (as an elongation mark) in the mouse E13.5 fetal liver as a source of primary definitive erythroid cells. At promoters of ectopically expressed genes, where only Nan-EKLF (but not WT) binding is expected, we predominantly find increased levels of both paused and elongating RNA Pol II suggesting that Nan-EKLF binding activates transcription at ectopic genes by RNA Pol II recruitment and promoter proximal pausing. Further, we find increased levels of H3K27ac and CBP occupancy at these sites indicating that the mechanism of Pol II recruitment relies on CBP-mediated H3K27 acetylation and increased chromatin accessibility. Overall, this suggests robust pioneering activity of Nan-EKLF likely mediated by the interaction of its TAD with the CBP/p300 acetylase complex.At genes downregulated in Nan/+ we find two major patterns of Pol II occupancy. One is the converse of that seen at ectopic genes wherein there is a concomitant decrease in both Pol II p-Ser5 and p-Ser2 levels, along with lower H3K27ac and CBP levels suggesting EKLF gene activation has been lost at these sites in Nan/+. This includes cell cycle EKLF targets such as E2f2 and Rgcc. The second set of genes have comparable levels of p-Ser5 (paused) Pol II in Nan/+ and WT, but lower levels of p-Ser2 (elongating) Pol II in Nan/+. This suggests that although Pol II is being recruited to the TSS and pauses effectively, the pause-release step leading to effective transcription elongation is impaired. This subset includes important EKLF targets such as Bcl11a, Pax7, Xpo7, and several membrane transporters. As expected, CBP and H3K27ac levels are similar in WT and Nan/+ at these sites.To determine the cause of impaired RNA Pol II pause-release we examined the global occupancies of key transcription elongation factors such as P-TEFb and NELF. We find that levels of NELF, a negative elongation factor, remain unchanged in WT and Nan/+. However, levels of the P-TEFb subunit Cdk9, a positive elongation factor that facilitates release of paused RNA Pol II, is significantly lower at the TSS of these genes in Nan/+. This suggests that in Nan/+, possible reduction or loss of EKLF binding at some EKLF target promoters impairs effective recruitment of positive transcription elongation factors, resulting in a failure to efficiently release paused RNA Pol II. This causes downregulation of these EKLF target genes and contributes to the severe anemic phenotypes of the Nan mouse.We conclude that: EKLF exhibits expression control of its target genes at both the transcriptional initiation and elongation steps in vivo; EKLF can act as a pioneer transcription factor and increase chromatin accessibility through H3K27 acetylation by CBP leading to recruitment and pausing of RNA Pol II; and EKLF recruits the positive transcription elongation complex P-TEFb, enabling the controlled release of paused RNA Pol II at transcription start sites of a select group of its targets. DisclosuresNo relevant conflicts of interest to declare.