Stem Cell Model of Novel RPL30 Variant in Diamond Blackfan Anemia with Downregulated GATA1-HSP70 in Early Erythroid Progenitors
Authors: Prosser Alexandra , Pushel Irina , Seidel Chris , Nemechek Jacqelyn , Leyda Molly , Prem Kumar Priyanka , Vivian Jay , Gerton Jennifer , Perry John M. 
Published: December 2024

Introduction:
Diamond Blackfan anemia (DBA) is caused by ribosomal protein gene mutations leading to increased apoptosis of erythroid progenitors. We identified a novel heterozygous variant (c.167+769C&gt;T) in the noncoding region of RPL30 in a patient diagnosed with DBA. RPL30 variants have not been reported in DBA, although the gene is predicted to be intolerant to loss of function. We hypothesized that this variant would negatively impact ribosomal biogenesis, specifically in early erythroid progenitors.
Methods:
To study the role of our novel variant, we developed an induced pluripotent stem cell (iPSC) model, including wild type (WT) and CRISPR-Cas9 edited RPL30 mutant clones. iPSCs were differentiated into hematopoietic stem cells, which were phenotypically confirmed by flow cytometry and functionally confirmed by colony forming unit (CFU) assays. Single cell RNA sequencing (scRNAseq) identified erythroid clusters for differential gene expression (DGE) analysis, using R Studio DESeq followed by Gene Ontology (GO) enrichment analysis.
Results:
Compared to WT, RPL30 mutant cells had significantly decreased expression of RPL30 (log2 fold change (FC) 0.53, adjusted p-value (p adj) 1.1e-4), potentially due to alternative splicing. The top 20 differentially expressed genes revealed downregulation of HSPA1A (log2 FC -1.8, p adj 1.1e-21) which encodes heat shock protein 70 (HSP70). HSP70 protein chaperones erythroid transcription factor GATA1 to avoid caspase 3 cleavage during differentiation. Loss of HSP70 protein has been implicated in RPL5 and RPL11 mutant erythroid cells previously as a potential modulator of severe DBA phenotype, although changes in transcription in early erythroid progenitors as seen here has not been described. DGE analysis also demonstrated downregulation ofPKIB (log2FC -2.0, p adj 8.6e-23), involved in stimulating hematopoietic differentiation, and upregulation of NLRC5 (log2FC 1.6, p adj 3.4e-22), innate immune and heme sensor as well as cell death regulator. Upon GO enrichment analysis of downregulated genes in RPL30 mutants, biologic process terms GO:0042254 ribosome biogenesis (p adj 1.3e-23), GO:1903708 positive regulation of hemopoiesis (p adj 9.1e-5), and GO:0045646 regulation of erythrocyte differentiation (p adj 4.2e-2) were all highlighted as driver terms.
Conclusions:
These results support our hypothesis that the RPL30 variant has a cellular impact that involves downregulation of ribosome biogenesis and erythropoiesis. HSPA1A downregulation suggests a potential role of HSP70 protein in our mutant model, reflecting previous RPL studies. Future directions include CFU assays for functional evaluation of erythropoiesis in the RPL30 mutants as well as molecular studies of these top differentially expressed genes. Our model will help demonstrate the role of RPL30 in DBA pathogenesis as well as provide new understanding of molecular pathways driving this disease, such as HSP70, in the setting of RPL mutations.