Functional impairment of erythropoiesis in Congenital Dyserythropoietic Anaemia type I arises at the progenitor level

The question of why certain cell types are differentially susceptible to mutations in broadly expressed genes remains largely unanswered. An important first step in determining the vulnerability of a specific lineage is to identify at what stage its requirement for a given protein arises. Strikingly, many types of congenital anaemia result from mutations in widely, if not ubiquitously, expressed proteins involved in core cellular processes. Examples include: mutations in ribosomal proteins causing Diamond Blackfan Anaemia1; mutations in the secretory vesicle protein SEC23B causing Congential Dyserythropoietic Anaemia (CDA) type II2; in CDA type III, arising from mutations in the kinesin encoded by KIF23 and RACGAP13, 4; and CDA type I (CDA-I) caused by mutations in CDAN1 or CDIN1.5 CDA-I is an example of a rare disease which has the potential to inform us about general cellular processes. CDIN1 and CDAN1 are widely expressed and loss of either protein is incompatible with life; however, only developing erythroblasts appear to be susceptible to biallelic hypomorphic mutations in either gene. We have recently shown that ex vivo differentiated erythroid cells from patients with CDA-I are delayed during terminal erythroid differentiation and this is associated with increased proliferation and widespread changes in chromatin accessibility.6 However, these studies were performed on CD34+ cells, which represent a mixture of haematopoietic stem and progenitor cells (HSPCs). The aim of this study was to investigate the precise cellular stage affected in CDA-I by assaying the functional consequences of CDAN1/CDIN1 mutations (Table 1) on the frequency and erythroid output of myeloid progenitors in patients with CDA-I. We show CDA-I does not alter the myeloid compartment; however, the functional output of CDA-I megakaryocyte and erythroid progenitors (MEPs) is compromised. Both in clonogenic assays and in liquid culture, CDA-I MEPs produce a higher proportion of aberrant colonies and show reduced expression of the erythroid lineage markers CD235 and CD71. c.1104_1106delCTT c.3128A > T p.Phe369del p.Asp1043Val c.2015C > T c.2681_2682delAG Venesection IFN-α2a c.2015C > T c.3338 T > C p.Pro672Leu p.Leu1113Pro c.2044C > T c.2744_2767del Whether the erythroid defect in CDA-I is confined to terminally maturing erythroblasts or also affects their progenitors remains an open question. To address this, we first investigated the expression of both causative genes (CDAN1 and CDIN1) in primary human HSPCs from healthy individuals. We found that similar expression levels of both genes were maintained throughout the haematopoietic hierarchy from HSC through to committed myeloid and erythroid progenitors, showing a decrease during terminal erythroid differentiation from the basophilic erythroblast stage onwards (Figure 1A, B).9 Next, to determine when defects first arise in CDA-I erythropoiesis, we compared the frequency of the progenitor cells upstream of erythroblasts in the haematopoietic hierarchy [Common Myeloid Progenitors (CMP), Megakaryocyte-Erythroid Progenitors (MEPs) and Granulocyte-Macrophage Progenitors (GMPs)] in peripheral blood (PB) of patients with CDA-I and controls (Figure 1C). This showed that the frequency of all three progenitor cell types was the same in CDA-I patients (n = 5) compared to controls (n = 17) (Figure 1C), indicating that biallelic mutations in CDAN1 or CDIN1 have no effect on the numbers of myeloid progenitors with erythroid potential, despite expression of both genes in progenitors (Figure 1B). This finding is in contrast to the situation in Diamond Blackfan Anaemia (DBA), where there are quantitative and qualitative abnormalities in erythroid progenitors.10 To assess whether the function of these myeloid progenitors was affected by biallelic mutations in CDAN1 or CDIN1, we sorted individual MEPs from healthy individuals (n = 10) and CDA-I patients (n = 5) into methylcellulose and cultured them for 14 days. The overall plating efficiency and proportion of MEPs that gave rise to erythroid colonies was similar in patients with CDA-I and controls ~70% (Figure 1D). However, whilst 100% of the erythroid output from control MEPs produced mature BFU-Es, 16.9% ± 3.7 of the erythroid colonies produced by CDA-I MEPs were small, poorly haemoglobinised erythroid clusters that were not seen in the MEP cultures from healthy donors (Figure 1D). These data suggest that, in patients with CDA-I, mutations in CDAN1 or CDIN1 cause perturbation of erythropoiesis at an earlier stage of erythroid differentiation (MEP) than previously reported, with samples from patients with mutations in either gene being similarly affected. Consistent with this, in three CDA-I patients with CDAN1 mutations, treatment with IFN-α2a, the only current therapy for CDA-I,5 restored the normal frequency and pattern of MEP-derived erythroid colonies (Figure 1D) and also normalized Hb levels in patients (Figure 1E). Sorting of individual MEPs into liquid culture, to allow more terminal differentiation, confirmed a qualitative defect in the number of MEP derived colonies grown from CDA-I MEPs (n = 36 colonies) compared to controls (n = 62 colonies) (Figure S1a). Meanwhile, interestingly, MEPs (n = 91) cultured from three CDA-I patients who were on IFN-α2a treatment at the time of sampling, showed normal erythroid maturation in vitro with only 6% aberrant colonies compared to 36% from the untreated CDA-I patients (Figure S1b). In summary, we provide evidence that defects in erythroid development in the rare inherited anaemia CDA-I arise at the MEP stage of erythroid differentiation, much earlier than previously recognized. There is no reported effect on the megakaryocytic lineage and platelet counts in CDA-I patients appear to be normal (Table 1). In common with other cell types, the question of why megakaryocytes are unaffected in CDA-I remains unclear. One explanation may be functional redundancy, as is presumed to be the case in other cell types. Some insight into this may come from analysis of platelet RNA, which suggests that megakaryocytes express a higher level of CDIN1 than CDAN1, the latter of which is below the level of detection.11 Our data also suggest that the erythroid defect can be corrected by IFN-α2a. It remains to be shown whether abnormal MEPs in CDA-I give rise exclusively to abnormal erythroblasts. Current understanding suggests the proteins encoded by CDIN1 and CDAN1 play a role in DNA repair and/or chromatin assembly. Analysing erythroid cells harbouring mutations in both genes would be likely to elucidate this novel pathway, however, generating suitable models will be technically challenging. By refining the stages of erythroid differentiation affected in CDA-I, this study paves the way for further work to identify why erythroid progenitors at the MEP stage develop a specific requirement for the function of the proteins encoded by CDAN1 and CDIN1. To this end, single-cell multiomic approaches to characterize the molecular defect in CDA-I progenitors are ongoing in our laboratory. Such studies would have the potential to inform novel processes in normal and disease erythropoiesis. We thank the CDA-I patients for providing blood samples and the flow cytometry facility at the WIMM for providing cell analysis services and technical expertise. The authors declare no competing financial interests. CS extracted CD34+ cells from CDA-I patient blood and cones; CS and KB performed the flow cytometry analysis and sorting experiments with assistance from SC. CS and KB scored, imaged and flow cytometric analysis of colonies from the Methocult and liquid culture assays. IR, VJB, NR, CB, CS and KB conceived and designed experiments; NR provided conceptual advice and clinical oversight; CB created the figures; CS and CB wrote the paper and all authors reviewed and critically edited the manuscript. RB, QAH, SO, RR, KR, MC and NR are the clinicians responsible for the care of the CDA-I patients. Raw RNAseq data from Gene Expression Omnibus (GEO) GSE74912 and GSE115684 was analysed in Figure 1B. FigS 1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.