Single Amino Acid Mutation of Rps19 Leading to Hematopoietic Stem and Progenitor Cells Defects in the Mouse Model of Diamond-Blackfan Anemia

Introduction: Although many underlying mutations in the ribosomal protein S19 (RPS19), a component of the small 40S ribosomal subunit, have been identified, their mechanistic consequences are not well understood. RPS19 is by far the most mutated gene involved in the pathology of Diamond-Blackfan anemia (DBA) (<25%), a rare bone marrow failure syndrome with reduced erythroid lineage development and early onset with lifelong consequences. DBA is often associated with low birth weight, generalized growth retardation and short stature, but is also accompanied by a diverse spectrum of cardiovascular, skeletal and urogenital abnormalities. Previously described Rps19 mutations in mice exhibited a spectrum of phenotypes commonly associated with DBA but lacked craniofacial, skeletal and other developmental abnormalities.

Methods: We have created a novel transgenic mouse model that carries single amino acid deletion of conserved arginine 67 (Rps19^R67∆) located in a frequently mutated DBA hot-spot, a post-translational modification site of the protein-arginine methyltransferase (Prmt) family of enzymes, which was removed by transcription activator of effector nuclease (TALEN)-specific mutagenesis. Although a compensatory role for loss-of-function of Trp53 in the DBA phenotype has already been established, the introduction of a homozygous Trp53 deletion in the Rps19^{R67∆/R67∆} mouse background provided a basis for studying the genetic interaction and phenotypic compensation between the ribosomal protein Rps19 and the transcription factor Trp53 in a double homozygous mouse model. We performed numerous phenotypic measurements and functional assays in primary hematopoietic organs, including bulk and single-cell transcriptomic profiling of erythroid-oriented hematopoietic cells and progenitors from fetal E14.5 liver and 10-week-old bone marrow.

Results: The homozygous deletion of the conserved arginine 67 in mouse resulted in delayed growth (~25% lower weight gain), shorter lifespan (median survival 20 weeks) and anemia (preceded by reduced proerythroblast and basophilic erythroblast counts in bone marrow and spleen) combined with severe cardiovascular (right atrial and ventricular hypertrophy, dystrophic diaphragm), urogenital (hypoplastic kidneys), skeletal (abnormalities of cervical and thoracic vertebral column), craniofacial (lack of ossification in the frontal bones and absence of closure in some of the cranial sutures), and cerebral (hydrocephalus) abnormalities with variable penetrance. Some of these abnormalities are already present in utero as E14.5-E18.5 Rps19^{R67∆/R67∆} fetuses are smaller, have smaller fetal livers, heart & kidney hypoplasia, and cerebral malformations.

The decreased erythroid lineage development in fetal liver as well as bone marrow was also characterized by upregulation of Trp53 signaling pathway in erythroid-biased hematopoietic stem cells and progenitors and relative enrichment of bone marrow progenitor populations (c-Kit+, Sca1+). Competitive transplantation assays using Rps19-deficient bone marrow cells indicated that HSCs and their descendants were compromised even if transplanted in excess (25% reconstitution vs. 75% controls). This defect, however, may be rescued by concomitant homozygous deletion of tumor suppressor Trp53 and double homozygous Rps19^{R67∆/R67∆} Trp53−/− hematopoietic cells achieve competitive advantage comparable to Trp53-/- mutants. Contrary to Rps19 mutants, the double mutant mice normally developped without DBA malformations, nevertheless their life-span was reduced due to the underlying Trp53 deficiency (median lifespan of 25 weeks). Transcriptomic analyses of hematopoietic stem and progenitor cells from bone marrow and E14.5 fetal liver of Rps19^{R67∆/R67∆} mutants identified a highly selective group of p53 transcriptional targets revalidating the role of augmented p53 signaling in the pathology of DBA.

Conclusion: Overall, our work represents a novel mouse model for DBA research and appears to be a new and very useful tool to further study the molecular pathology of complex multisystem failure in DBA and to design new therapeutic options for DBA. Currently, our analysis shows that some p53 targets may be mediators of the DBA phenotype and their therapeutic interference is the focus of our research.

No relevant conflicts of interest to declare.