Presenter Status
Fellow
Abstract Type
Research
Primary Mentor
John Perry
Start Date
15-5-2025 12:45 PM
End Date
15-5-2025 1:00 PM
Presentation Type
Oral Presentation
Description
Background: Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome caused by ribosomal protein gene mutations leading to apoptosis of erythroid progenitors. We identified a novel heterozygous variant (c.167+769C>T) in the noncoding region of RPL30 in a patient diagnosed with DBA.
Objectives/Goal: We hypothesized that this variant stunts erythroid differentiation at the proerythroblast stage and is pathogenic for DBA.
Methods/Design: We developed an induced pluripotent stem cell (iPSC) model with a wild type (WT) and three CRISPR-Cas9 edited RPL30 mutant clones. iPSCs were differentiated into hematopoietic stem cells, which were assessed by flow cytometry and single cell RNA sequencing (scRNAseq, analysis with R Studio DESeq). Erythroid differentiation with specialized cell culture techniques is being performed with plans for functional assessment of colonies, hemoglobin staining, and flow cytometry.
Results: As hematopoietic stem cells, the RPL30 mutant clones demonstrated increased early erythroid progenitors and decreased later differentiated erythroid cells compared to WT (CD235+CD71+CD34+CD105+ proerythroblasts averaged 96.3% compared to 81% while CD235+CD71+CD34-105+/- basophilic-polychromatic-orthochromatic erythroblasts averaged 1.3% compared to 14.3%) . With scRNAseq, 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. With downregulated HSP70, increased GATA1 protein degradation is predicted which is supported by downregulation of GATA1 targets in our RPL30 mutant clones (HBA1, logFC 1.2, p adj 7.7e-18; HBA2, logFC 2.2, padj 2.2; HBB, logFC 1.1, padj 2.1e-3; ALAS2, logFC 1.6, padj 3.9e-9).
Conclusions: These early results support our hypothesis that the variant stunts erythroid differentiation and reveals the potential role of HSP70. HSP70 protein has been implicated in red blood cell disease previously and specifically in RPL5 and RPL11 mutant erythroid cells as a potential modulator of severe DBA phenotype, although changes in transcription in early erythroid progenitors as seen here has not been described. Our model will demonstrate the role of RPL30 in DBA pathogenesis as well as provide understanding of molecular pathways driving this disease, such as HSP70, in the setting of RPL mutations.
Included in
Biological Phenomena, Cell Phenomena, and Immunity Commons, Genetic Phenomena Commons, Hematology Commons, Higher Education and Teaching Commons, Medical Education Commons, Pediatrics Commons, Science and Mathematics Education Commons
Novel RPL30 variant in Diamond Blackfan anemia demonstrates early impact on erythroid differentiation with downregulated GATA1-HSP70
Background: Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome caused by ribosomal protein gene mutations leading to apoptosis of erythroid progenitors. We identified a novel heterozygous variant (c.167+769C>T) in the noncoding region of RPL30 in a patient diagnosed with DBA.
Objectives/Goal: We hypothesized that this variant stunts erythroid differentiation at the proerythroblast stage and is pathogenic for DBA.
Methods/Design: We developed an induced pluripotent stem cell (iPSC) model with a wild type (WT) and three CRISPR-Cas9 edited RPL30 mutant clones. iPSCs were differentiated into hematopoietic stem cells, which were assessed by flow cytometry and single cell RNA sequencing (scRNAseq, analysis with R Studio DESeq). Erythroid differentiation with specialized cell culture techniques is being performed with plans for functional assessment of colonies, hemoglobin staining, and flow cytometry.
Results: As hematopoietic stem cells, the RPL30 mutant clones demonstrated increased early erythroid progenitors and decreased later differentiated erythroid cells compared to WT (CD235+CD71+CD34+CD105+ proerythroblasts averaged 96.3% compared to 81% while CD235+CD71+CD34-105+/- basophilic-polychromatic-orthochromatic erythroblasts averaged 1.3% compared to 14.3%) . With scRNAseq, 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. With downregulated HSP70, increased GATA1 protein degradation is predicted which is supported by downregulation of GATA1 targets in our RPL30 mutant clones (HBA1, logFC 1.2, p adj 7.7e-18; HBA2, logFC 2.2, padj 2.2; HBB, logFC 1.1, padj 2.1e-3; ALAS2, logFC 1.6, padj 3.9e-9).
Conclusions: These early results support our hypothesis that the variant stunts erythroid differentiation and reveals the potential role of HSP70. HSP70 protein has been implicated in red blood cell disease previously and specifically in RPL5 and RPL11 mutant erythroid cells as a potential modulator of severe DBA phenotype, although changes in transcription in early erythroid progenitors as seen here has not been described. Our model will demonstrate the role of RPL30 in DBA pathogenesis as well as provide understanding of molecular pathways driving this disease, such as HSP70, in the setting of RPL mutations.
