RPS26-haploinsufficient patient-derived iPSCs exhibit impaired processing of ribosomal RNA and upregulate ribosome biogenesis factors.
Diamond-Blackfan Anemia Syndrome (DBAS) is characterized by hypoplastic anemia, congenital malformations and cancer predisposition, and is predominantly caused by ribosomal protein gene mutations, including RPS26 in ~10% of patients. Pathogenic mechanisms underlying RPS26-DBAS are largely unknown. To address this, we established induced pluripotent stem cell (iPSC) models derived from patients with RPS26 mutations to study defects in ribosome biogenesis and hematopoiesis.
We generated iPSC lines from a patient with RPS26-DBAS from the Dutch registry. Erythroblasts were reprogrammed using the Cytotune-Sendai-IPS2.0 kit. Using CRISPR/Cas9-mediated homology-directed repair, we corrected the RPS26 mutation in the patient iPSC lines. We compared control, mutant and corrected lines through differentiation into embryoid bodies (EB), hematopoietic organoids, and hematopoietic progenitors. All lines were characterized by a multi-omics approach.
Whole genome sequencing confirmed successful correction of the RPS26 mutation. Mutant lines exhibited disaggregation of iPSC-derived EBs and failed hematopoietic organoid formation, while corrected lines showed restored EB integrity and hematopoietic development. Pre-ribosomal-RNA profiling revealed accumulation of 26S and 18S-E pre-ribosomal RNA in mutant lines, consistent with RPS26 haploinsufficiency, which was fully reverted in corrected lines. Proteomic analysis demonstrated increased expression of ribosomal biogenesis factors, including PNO1, in mutant lines. Increased expression was not determined with RNA sequencing, suggesting a distinct protein translation signature in RPS26-mutant cells, compared to corrected and control cells.
Our data demonstrate that patient-iPSC lines represent valid models to study DBAS-disease mechanisms. Molecular characterization of our RPS26-DBAS lines elucidated key proteins involved in ribosomal processing that contribute to a unique RPS26-associated ribosomopathy.