Deficiency of Lipin2 Results in Enhanced NF-κB Signaling and Osteoclast Formation in RAW-D Murine Macrophages.

Lipin2 is a phosphatidate phosphatase that plays critical roles in fat homeostasis. Alterations in Lpin2, which encodes lipin2, cause the autoinflammatory bone disorder Majeed syndrome. Lipin2 limits lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. However, little is known about the precise molecular mechanisms underlying its anti-inflammatory function. In this study, we attempted to elucidate the molecular link between the loss of lipin2 function and autoinflammatory bone disorder. Using a Lpin2 knockout murine macrophage cell line, we showed that lipin2 deficiency enhances innate immune responses to LPS stimulation through excessive activation of the NF-κB signaling pathway, partly because of TAK1 signaling upregulation. Lipin2 depletion also enhanced RANKL-mediated osteoclastogenesis and osteoclastic resorption activity accompanied by NFATc1 dephosphorylation and increased nuclear accumulation. These results suggest that lipin2 suppresses the development of autoinflammatory bone disorder by fine-tuning proinflammatory responses and osteoclastogenesis in macrophages. Therefore, this study provides insights into the molecular pathogenesis of monogenic autoinflammatory bone disorders and presents a potential therapeutic intervention.

Majeed Syndrome: A Review of the Clinical, Genetic and Immunologic Features.

Majeed syndrome is a multi-system inflammatory disorder affecting humans that presents with chronic multifocal osteomyelitis, congenital dyserythropoietic anemia, with or without a neutrophilic dermatosis. The disease is an autosomal recessive disorder caused by mutations in LPIN2, the gene encoding the phosphatidic acid phosphatase LIPIN2. It is exceedingly rare. There are only 24 individuals from 10 families with genetically confirmed Majeed syndrome reported in the literature. The early descriptions of Majeed syndrome reported severely affected children with recurrent fevers, severe multifocal osteomyelitis, failure to thrive, and marked elevations of blood inflammatory markers. As more affected families have been identified, it has become clear that there is significant phenotypic variability. Data supports that disruption of the phosphatidic acid phosphatase activity in LIPIN2 results in immune dysregulation due to aberrant activation of the NLRP3 inflammasome and overproduction of proinflammatory cytokines including IL-1ß, however, these findings did not explain the bone phenotype. Recent studies demonstrate that LPIN2 deficiency drives pro-inflammatory M2-macrophages and enhances osteoclastogenesis which suggest a critical role of lipin-2 in controlling homeostasis at the growth plate in an inflammasome-independent manner. While there are no approved medications for Majeed syndrome, pharmacologic blockade of the interleukin-1 pathway has been associated with rapid clinical improvement.

Genetic and functional insights into CDA-I prevalence and pathogenesis.

BACKGROUND: Congenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes CDAN1 and C15orf41. Little is understood about either protein and it is unclear in which cellular pathways they participate. METHODS: Genetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by CDAN1. Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation. RESULTS: We identify six novel CDAN1 mutations and one novel mutation in C15orf41 and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells. CONCLUSION: Stability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.

VPS4A Mutations in Humans Cause Syndromic Congenital Dyserythropoietic Anemia due to Cytokinesis and Trafficking Defects.

The Congenital Dyserythropoietic Anemia (CDA) Registry was established with the goal to facilitate investigations of natural history, biology, and molecular pathogenetic mechanisms of CDA. Three unrelated individuals enrolled in the registry had a syndrome characterized by CDA and severe neurodevelopmental delay. They were found to have missense mutations in VPS4A, a gene coding for an ATPase that regulates the ESCRT-III machinery in a variety of cellular processes including cell division, endosomal vesicle trafficking, and viral budding. Bone marrow studies showed binucleated erythroblasts and erythroblasts with cytoplasmic bridges indicating abnormal cytokinesis and abscission. Circulating red blood cells were found to retain transferrin receptor (CD71) in their membrane, demonstrating that VPS4A is critical for normal reticulocyte maturation. Using proband-derived induced pluripotent stem cells (iPSCs), we have successfully modeled the hematologic aspects of this syndrome in vitro, recapitulating their dyserythropoietic phenotype. Our findings demonstrate that VPS4A mutations cause cytokinesis and trafficking defects leading to a human disease with detrimental effects to erythropoiesis and neurodevelopment.

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

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

A Very Rare Congenital Dyserythropoietic Anemia Variant-Type IV in a Patient With a Novel Mutation in the KLF1 Gene: A Case Report and Review of the Literature.

Congenital dyserythropoietic anemias comprise a group of very rare hereditary disorders characterized by ineffective erythropoiesis and distinct morphologic abnormalities of the erythroblasts in the bone marrow. The wide variety of phenotypes observed in these patients makes the diagnosis difficult; identification of the genetic variants is crucial in differential diagnosis and clinical management. We report the nineth case with congenital dyserythropoietic anemia type IV, with a novel mutation that has not been reported before.

Hematopoietic Stem Cell Transplantation in Congenital Dyserythropetic Anemia Type II: A Case Report and Review of the Literature.

Currently, there is no guideline for the treatment of patients with congenital dyserythropoietic anemia (CDA) type II. One approach is to follow-up patients with transfusions, on the basis of individually determined target hemoglobin levels, and iron chelation according to the thalassemia guidelines. In some transfusion-dependent CDA II patients, splenectomy reduces the number of transfusions; however, the only known curative option for CDA II patients is hematopoietic stem cell transplantation (HSCT). Only a few published case reports of allogeneic HSCT in CDA II patients are available. Here, we review the literature and add our data of a CDA II patient who developed transfusion dependence and was cured with HSCT.

KLF1 mutation E325K induces cell cycle arrest in erythroid cells differentiated from congenital dyserythropoietic anemia patient-specific induced pluripotent stem cells.

Krüppel-like factor 1 (KLF1), a transcription factor controlling definitive erythropoiesis, is involved in sequential control of terminal cell division and enucleation via fine regulation of key cell cycle regulator gene expression in erythroid lineage cells. Type IV congenital dyserythropoietic anemia (CDA) is caused by a monoallelic mutation at the second zinc finger of KLF1 (c.973G>A, p.E325K). We recently diagnosed a female patient with type IV CDA with the identical missense mutation. To understand the mechanism underlying the dyserythropoiesis caused by the mutation, we generated induced pluripotent stem cells (iPSCs) from the CDA patient (CDA-iPSCs). The erythroid cells that differentiated from CDA-iPSCs (CDA-erythroid cells) displayed multinucleated morphology, absence of CD44, and dysregulation of the KLF1 target gene expression. In addition, uptake of bromodeoxyuridine by CDA-erythroid cells was significantly decreased at the CD235a+/CD71+ stage, and microarray analysis revealed that cell cycle regulator genes were dysregulated, with increased expression of negative regulators such as CDKN2C and CDKN2A. Furthermore, inducible expression of the KLF1 E325K, but not the wild-type KLF1, caused a cell cycle arrest at the G1 phase in CDA-erythroid cells. Microarray analysis of CDA-erythroid cells and real-time polymerase chain reaction analysis of the KLF1 E325K inducible expression system also revealed altered expression of several KLF1 target genes including erythrocyte membrane protein band 4.1 (EPB41), EPB42, glutathione disulfide reductase (GSR), glucose phosphate isomerase (GPI), and ATPase phospholipid transporting 8A1 (ATP8A1). Our data indicate that the E325K mutation in KLF1 is associated with disruption of transcriptional control of cell cycle regulators in association with erythroid membrane or enzyme abnormalities, leading to dyserythropoiesis.

Genetic disarray follows mutant KLF1-E325K expression in a congenital dyserythropoietic anemia patient.

Congenital dyserythropoietic anemia type IV is caused by a heterozygous mutation, Glu325Lys (E325K), in the KLF1 transcription factor. Molecular characteristics of this disease have not been clarified, partly due to its rarity. We expanded erythroid cells from a patient's peripheral blood and analyzed its global expression pattern. We find that a large number of erythroid pathways are disrupted, particularly those related to membrane transport, globin regulation, and iron utilization. The altered genetics lead to significant deficits in differentiation. Glu325 is within the KLF1 zinc finger domain at an amino acid critical for site specific DNA binding. The change to Lys is predicted to significantly alter the target site recognition sequence, both by subverting normal recognition and by enabling interaction with novel sites. Consistent with this, we find high level ectopic expression of genes not normally present in the red cell. These altered properties explain patients' clinical and phenotypic features, and elucidate the dominant character of the mutation.

Fetal-onset Congenital Dyserythropoietic Anemia Type 1 due to a Novel Mutation With Severe Iron Overload and Severe Cholestatic Liver Disease.

We report a rare case of severe congenital dyserythropoietic anemia type 1 with fetal onset. Our patient presented with fetal hydrops from 19 weeks of gestation, requiring multiple intrauterine transfusions. At birth, she had severe hemolytic anemia with severe jaundice, and was subsequently transfusion dependent. She eventually developed severe iron overload and fulminant liver failure before her demise at 5 months of age. Genetic testing revealed a novel mutation in CDAN1.

KLF1 E325K-associated Congenital Dyserythropoietic Anemia Type IV: Insights Into the Variable Clinical Severity.

We identified a child with KLF1-E325K congenital dyserythropoietic anemia type IV who experienced a severe clinical course, fetal anemia, hydrops fetalis, and postnatal transfusion dependence only partially responsive to splenectomy. The child also had complete sex reversal, the cause which remains undetermined. To gain insights into our patient's severe hematologic phenotype, detailed analyses were performed. Erythrocytes from the patient and parents demonstrated functional abnormalities of the erythrocyte membrane, attributed to variants in the α-spectrin gene. Hypomorphic alleles in SEC23B and YARS2 were also identified. We hypothesize that coinheritance of variants in relevant erythrocyte genes contribute to the clinical course in our patient and other E325K-linked congenital dyserythropoietic anemia IV patients with severe clinical phenotypes.