Presenter Status
Fellow
Abstract Type
Research
Primary Mentor
Jennifer Gerton
Start Date
11-5-2023 11:30 AM
End Date
11-5-2023 1:30 PM
Presentation Type
Abstract
Description
Background: Bone marrow failure is life-threatening and requires prompt, intensive treatment, which is dependent upon identification of genetic drivers. Although there has been investigation into associated genetic mutations, especially in Diamond Blackfan anemia (DBA), the relationship between genotype and phenotype remains unclear.DBA has been identified as a ribosomopathy, also known as a disease associated with defects in ribosome biogenesis. We identified a novel heterozygous variant (c.167+769C >T) in the noncoding region of RPL30 in a patient with clinical diagnosis of DBA. Clinical RNA sequencing (RNA-seq) suggests the variant generates a novel splice acceptor site resulting in truncated RPL30 transcripts. Elucidation of the function of this variant along with its relationship with DBA will provide new insight into the pathogenesis of disease.
Objectives/Goal: Our central hypothesis is that our RPL30 variant leads to insufficient levels of ribosomal protein L30, which induces compromised ribosome assembly in the nucleolus, protein synthesis, and ultimately abnormal hematopoietic differentiation. For this stage of the project, we are completing preliminary work to establish a human pluripotent stem cell line (hiPSC) with the variant, investigating its impact on transcription and production of ribosomal protein, and validating hematopoietic differentiation methods.
Methods/Design: For our cell line model, the Stowers Institute Genomic Engineering core facility utilized CRISPR-Cas9 to generate the RPL30 variant in a differentiated human cell line (retinal pigment epithelial cell line or RPE1). We have four homozygous clones in RPE1 that we are utilizing for preliminary testing and are awaiting our hiPSC line, which is currently in development. We have utilized western blot to assess protein levels and are applying RNA-seq to the variant clones to examine RPL30 transcript levels for the expected splicing defect. We have also optimized a hematopoietic differentiation protocol with wild type hiPSC line. We performed flow cytometry to evaluate cell surface markers of hematopoietic progenitors and established a gating strategy, ready for application to the variant hiPSC line.
Results: With the established RPE1 variant clones, we have consistently detected L30 protein and plan for further quantification to compare levels of protein. We have effectively isolated RNA from wild type and RPE1 variant clones and have submitted to the Stowers Sequencing core facility for RNA sequencing. For hematopoietic differentiation of wild type hiPSC, we have evaluated clusters of differentiation (CD) markers on Days 14 and 20 of differentiation that are consistent with previously published values.
Conclusions: We are completing preliminary work needed for this project. We anticipate having the hiPSC line ready soon along with RNA-seq and protein quantification data. For further optimization of our differentiation approach, we will apply a colony forming assay for wild type. For next stages, we will determine the functional impact of this variant by applying the hematopoietic differentiation protocol to the variant cell line, utilizing microscopy to evaluate nucleolar morphology, and performing multiple techniques to assess ribosome production, translation, and signaling pathway activity. Long term, we expect to inform the study of ribosomopathies, create a template for future studies, and positively impact the understanding of pediatric bone marrow failure.
MeSH Keywords
anemia, Diamond-Blackfan; bone marrow failure syndromes; large ribosome subunit; hematopoiesis; alternative splicing; induced pluripotent stem cells
Functional evaluation of a novel RPL30 mutation and its role in Diamond Blackfan anemia (DBA)
Background: Bone marrow failure is life-threatening and requires prompt, intensive treatment, which is dependent upon identification of genetic drivers. Although there has been investigation into associated genetic mutations, especially in Diamond Blackfan anemia (DBA), the relationship between genotype and phenotype remains unclear.DBA has been identified as a ribosomopathy, also known as a disease associated with defects in ribosome biogenesis. We identified a novel heterozygous variant (c.167+769C >T) in the noncoding region of RPL30 in a patient with clinical diagnosis of DBA. Clinical RNA sequencing (RNA-seq) suggests the variant generates a novel splice acceptor site resulting in truncated RPL30 transcripts. Elucidation of the function of this variant along with its relationship with DBA will provide new insight into the pathogenesis of disease.
Objectives/Goal: Our central hypothesis is that our RPL30 variant leads to insufficient levels of ribosomal protein L30, which induces compromised ribosome assembly in the nucleolus, protein synthesis, and ultimately abnormal hematopoietic differentiation. For this stage of the project, we are completing preliminary work to establish a human pluripotent stem cell line (hiPSC) with the variant, investigating its impact on transcription and production of ribosomal protein, and validating hematopoietic differentiation methods.
Methods/Design: For our cell line model, the Stowers Institute Genomic Engineering core facility utilized CRISPR-Cas9 to generate the RPL30 variant in a differentiated human cell line (retinal pigment epithelial cell line or RPE1). We have four homozygous clones in RPE1 that we are utilizing for preliminary testing and are awaiting our hiPSC line, which is currently in development. We have utilized western blot to assess protein levels and are applying RNA-seq to the variant clones to examine RPL30 transcript levels for the expected splicing defect. We have also optimized a hematopoietic differentiation protocol with wild type hiPSC line. We performed flow cytometry to evaluate cell surface markers of hematopoietic progenitors and established a gating strategy, ready for application to the variant hiPSC line.
Results: With the established RPE1 variant clones, we have consistently detected L30 protein and plan for further quantification to compare levels of protein. We have effectively isolated RNA from wild type and RPE1 variant clones and have submitted to the Stowers Sequencing core facility for RNA sequencing. For hematopoietic differentiation of wild type hiPSC, we have evaluated clusters of differentiation (CD) markers on Days 14 and 20 of differentiation that are consistent with previously published values.
Conclusions: We are completing preliminary work needed for this project. We anticipate having the hiPSC line ready soon along with RNA-seq and protein quantification data. For further optimization of our differentiation approach, we will apply a colony forming assay for wild type. For next stages, we will determine the functional impact of this variant by applying the hematopoietic differentiation protocol to the variant cell line, utilizing microscopy to evaluate nucleolar morphology, and performing multiple techniques to assess ribosome production, translation, and signaling pathway activity. Long term, we expect to inform the study of ribosomopathies, create a template for future studies, and positively impact the understanding of pediatric bone marrow failure.
