Gene expression profile of erythroblastic cells induced from congenital dyserythropoietic anemia (CDA) patient-derived induced pluripotent stem cells
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ABSTRACT: Analysis of erythroblastic cells differentiated from induced pluripotent stem cells (iPSCs) generated from peripheral blood T lymphocytes of CDA type IV patient. Type IV congenital dyserythropoietic anemia (CDA) is due to a monoallelic mutation at the second zinc finger of KLF1 (c.973G>A). Results provide insight into molecular mechanisms underlying CDA pathogenesis.
Project description:Red blood cell disorders can result in severe anemia. One such disease, congenital dyserythropoietic anemia IV (CDA IV) is caused by heterozygous mutation E325K in the transcription factor KLF1. However, studying the molecular basis of CDA IV is severely impeded by paucity of suitable and adequate quantities of material from anaemic patients and rarity of the disease. We therefore took a novel approach, creating a human cellular disease model system for CDA IV, which accurately recapitulates the disease phenotype. Next, using comparative proteomics we reveal extensive distortion of the proteome and a wide range of disordered biological processes in CDA IV erythroid cells. These include down-regulated pathways governing cell cycle, chromatin separation, DNA repair, cytokinesis, membrane trafficking and global transcription, and upregulated networks governing mitochondria biogenesis. The diversity of such pathways elucidates the spectrum of phenotypic abnormalities that occur with CDA IV and impairment to erythroid cell development and survival, collectively explaining the CDA IV disease phenotype. The data also reveal far more extensive involvement of KLF1 in previously assigned biological processes, along with novel roles in the regulation of intracellular processes not previously attributed to this transcription factor. Overall, the data demonstrate the power of such a model cellular system to unravel the molecular basis of disease and how studying effects of a rare mutation can reveal fundamental biology.
Project description:Congenital dyserythropoietic anaemia (CDA) type IV has been associated with an amino acid substitution, Glu325Lys (E325K), in the transcription factor KLF1. Patients with CDA type IV 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. To address this question, we generated iPSC lines genetically modified to express a KLF1-E325K-ERT2 protein that could be activated with 4OH-tamoxifen. We performed bulk RNA-sequencing on macrophages generated from these iPSCs, macrophages generated from one KLF1-E325K-ERT2 iPSC line (iCDA4.1) was compared to macrophages generated from one inducible KLF1-WT-ERT2 (K2) iPSC line which was derived from the same parental iPSCs (SFCi55) as the KLF1-E325K-ERT2 line.
Project description:Congenital dyserythropoietic anemia (CDA) type IV is caused by a heterozygous mutation, Glu325Lys (E325K), in the KLF1 transcription factor. A molecular understanding of this disease is absent, 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 the patients’ clinical and phenotypic features and elucidate the dominant character of the mutation.
Project description:Congenital Dyserythropoietic Anaemia type 1 (CDA-I) is an inherited anaemia arising primarily from mutations in C15orf41 and CDAN1, however the molecular mechanisms that cause the disease remain to be fully elucidated. We use an in vitro culture system to study multiple stages of erythropoiesis from CD34+ progenitors of patients with CDA-I. Applying a number of techniques, including ATAC-seq, we show that differentiation of CDA-I patient erythroblasts closely matches that of healthy donors during the early and intermediate stages of erythroid differentiation and maturation. However, a defect in terminal erythropoiesis can be observed in the CDA-I patient derived erythroblasts, resulting in a reduction in the number of enucleated erythroid cells.
Project description:To understand the fibrotic response in the CDA-HFD induced NASH fibrosis model, we performed RNA-seq on liver samples collected from mice fed with normal chow (week 0) or CDA-HFD chow (weeks 8 and 16).
Project description:We describe a case of severe neonatal anemia with kernicterus due to compound heterozygosity for null mutations in KLF1, each inherited from asymptomatic parents. One of the mutations is novel. This is the first described case of a KLF1 null human. The phenotype of severe DAT-negative non-spherocytic hemolytic anaemia (NSHA), jaundice, hepato-splenomegaly, and marked erythroblastosis is more severe than that present in CDA type IV due to dominant mutations in the second zinc-finger of KLF1. There was a very high level of HbF expression into childhood (>70%), consistent with a key role for KLF1 in human hemoglobin switching. We performed RNA-seq on circulating erythroblasts and found human KLF1 acts like mouse Klf1 to coordinate expression of many genes required to build a red cell including those encoding globins, cytoskeletal components, AHSP, heme synthesis enzymes, cell cycle regulators, and blood group antigens. We identify novel KLF1 target genes including KIF23 and KIF11 which are required for proper cytokinesis. We also identify new roles for KLF1 in autophagy, global transcriptional control and RNA splicing. We suggest loss of KLF1 should be considered in otherwise unexplained cases of severe neonatal NSHA or hydrops fetalis. mRNA sequencing on peripheral blood from a family trio (mother, father and proband) where parents were asymptomatic and proband had severe neonatal anemia.
Project description:We describe a case of severe neonatal anemia with kernicterus due to compound heterozygosity for null mutations in KLF1, each inherited from asymptomatic parents. One of the mutations is novel. This is the first described case of a KLF1 null human. The phenotype of severe DAT-negative non-spherocytic hemolytic anaemia (NSHA), jaundice, hepato-splenomegaly, and marked erythroblastosis is more severe than that present in CDA type IV due to dominant mutations in the second zinc-finger of KLF1. There was a very high level of HbF expression into childhood (>70%), consistent with a key role for KLF1 in human hemoglobin switching. We performed RNA-seq on circulating erythroblasts and found human KLF1 acts like mouse Klf1 to coordinate expression of many genes required to build a red cell including those encoding globins, cytoskeletal components, AHSP, heme synthesis enzymes, cell cycle regulators, and blood group antigens. We identify novel KLF1 target genes including KIF23 and KIF11 which are required for proper cytokinesis. We also identify new roles for KLF1 in autophagy, global transcriptional control and RNA splicing. We suggest loss of KLF1 should be considered in otherwise unexplained cases of severe neonatal NSHA or hydrops fetalis.