Modelling erythropoiesis in congenital dyserythropoietic anaemia type I (CDA-I)

Abstract

We employ and extensively characterise an ex vivo culture system to study terminal erythroid maturation of CD34 + progenitors from the peripheral blood of normal individuals and patients with Congenital Dyserythropoietic Anaemia type 1 (CDA-I). Using morphological analysis, FACS analysis and the proteomic approach CyTOF, we analysed patient-derived erythroblasts stage-matched with those from healthy donors during the expansion phase and into early differentiation. In patient cells, aspects of disordered erythropoiesis manifest midway through differentiation, including increased proliferation and changes in the DNA accessibility profile. We also show that cultured erythroblasts from CDA-I patients recapitulate the pathognomic feature of this erythroid disorder with up to 40% of the cells having abnormal 'spongy' chromatin morphology by electron microscopy, as well as upregulation of GDF15, a marker of ineffective erythropoiesis. In the tertiary phase of culture, patient cells show significantly less enucleation and there is persistence of earlier erythroid precursors. Furthermore, the enucleation defect appears to be more severe in patients with mutations in C15orf41, as compared to the other known causative gene CDAN1, indicating a genotype/phenotype correlation in CDA-I. Such erythroblasts are a valuable resource for investigating the pathogenesis of this disease and provide the opportunity for streamlining diagnosis for CDA-I patients and ultimately other forms of unexplained anaemia. Introduction:

An integrated platform to systematically identify causal variants and genes for polygenic human traits

“…Recently, ATAC-seq allowed a comprehensive identification of cis -regulatory elements which remain constitutively present or dynamically change throughout haematopoietic lineage specification, differentiation, and maturation 8,24 . To identify regulatory regions in early, intermediate and late erythroid cells we generated ATAC-seq from such cells obtained by ex vivo erythroid differentiation of CD34 + stem and progenitor cells 9 from three healthy, non-anaemic individuals (Supplementary Fig. 2).…”

“…Here, we have developed an integrated platform of experimental and computational methods to prioritise likely causal variants, link them to the genes they regulate, and determine the mechanism by which they alter gene function. To illustrate the approach we have initially focussed on a single haematopoietic lineage: the development of mature red blood cells (RBC), for which all stages of lineage specification and differentiation from a haematopoietic stem cell to a RBC are known, and can be recapitulated ex vivo by culture of CD34 + progenitor and stem cells [7][8][9] . GWASs have identified over 550 chromosome regions associated with changes in the phenotypes of mature RBC 10,11 ; within these regions 1,114 index SNPs are in high LD with 30,694 variants, of which, only eight have been claimed as causal regulatory variants through experimental validation [12][13][14][15][16] .…”

Genetic and functional insights into CDA-I prevalence and pathogenesis

“…Patients were assessed and analysed using the Oxford Red Cell Panel Targeted Resequencing strategy as previously described. 11 differentiation of Cd34 + cells CD34 + cells were isolated (as described in online supplementary methods) and differentiated using a modified version of a published three-phase protocol 12 or using a two-phase liquid culture. 13 14…”

“…We tested the effects of pathogenic mutations on Codanin-1 and C15orf41 by two colour near-infrared quantitative western blot using protein extracted from erythroblasts cultured from the peripheral blood of healthy individuals and patients with CDAN1 and C15orf41 CDA-I using a well characterised in vitro culture system 12 (figure 4B). To detect Codanin-1, we validated a polyclonal antibody from Bethyl Laboratories (catalogue number A304-951A) by showing it cross-reacts with Codanin-1 conjugated to mCherry when overexpressed in HEK293T cells (online supplementary figure 2A).…”

Direct Generation of Immortalized Erythroid Progenitor Cell Lines from Peripheral Blood Mononuclear Cells

“…Diamond Blackfan anemia (DBA) and congenital dyserythropoietic anemia (CDA) affect normal erythropoiesis due to deficiencies in important molecular processes in erythroid cells [ 2 , 3 ]. For understanding the molecular basis of normal versus pathological erythropoiesis, erythroid cells generated by in vitro erythropoiesis by differentiating primary CD34 + hematopoietic stem and progenitor cells (HSPCs) are commonly being used [ 4 , 5 , 6 , 7 , 8 , 9 ]. The main challenge in this approach is the requirement of multiple donations of HSPCs from a single individual to generate adequate numbers of erythroid cells for the downstream experiments.…”