SBDSR126T rescues survival of sbds -/- zebrafish in a dose-dependent manner independently of Tp53.

Defects in ribosomal biogenesis profoundly affect organismal development and cellular function, and these ribosomopathies produce a variety of phenotypes. One ribosomopathy, Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, pancreatic exocrine insufficiency, and skeletal anomalies. SDS results from biallelic mutations in SBDS, which encodes a ribosome assembly factor. Some individuals express a missense mutation, SBDS R126T , along with the common K62X mutation. We reported that the sbds-null zebrafish phenocopies much of SDS. We further showed activation of Tp53-dependent pathways before the fish died during the larval stage. Here, we expressed SBDS R126T as a transgene in the sbds -/- background. We showed that one copy of the SBDS R126T transgene permitted the establishment of maternal zygotic sbds-null fish which produced defective embryos with cdkn1a up-regulation, a Tp53 target involved in cell cycle arrest. None survived beyond 3 dpf. However, two copies of the transgene resulted in normal development and lifespan. Surprisingly, neutropenia persisted. The surviving fish displayed suppression of female sex differentiation, a stress response in zebrafish. To evaluate the role of Tp53 in the pathogenesis of sbds -/- fish phenotype, we bred the fish with a DNA binding deficient allele, tp53 M214K Expression of the loss-of-function tp53 M214K did not rescue neutropenia or survival in sbds-null zebrafish. Increased expression of cdkn1a was abrogated in the tp53 M214K/M214K ;sbds -/- fish. We conclude that the amount of SBDSR126T protein is important for development, inactivation of Tp53 fails to rescue neutropenia or survival in the sbds-null background, and cdkn1a up-regulation was dependent on WT tp53 We hypothesize that additional pathways are involved in the pathophysiology of SDS.

Effect of the unfolded protein response and oxidative stress on mutagenesis in CSF3R: a model for evolution of severe congenital neutropenia to myelodysplastic syndrome/acute myeloid leukemia.

Severe congenital neutropenia (SCN) is a rare blood disorder characterised by abnormally low levels of circulating neutrophils. The most common recurrent mutations that cause SCN involve neutrophil elastase (ELANE). The treatment of choice for SCN is the administration of granulocyte-colony stimulating factor (G-CSF), which increases the neutrophil number and improves the survival and quality of life. Long-term survival is however linked to the development of myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). About 70% of MDS/AML patients acquire nonsense mutations affecting the cytoplasmic domain of CSF3R (the G-CSF receptor). About 70% of SCN patients with AML harbour additional mutations in RUNX1. We hypothesised that this coding region of CSF3R constitutes a hotspot vulnerable to mutations resulting from excessive oxidative stress or endoplasmic reticulum (ER) stress. We used the murine Ba/F3 cell line to measure the effect of induced oxidative or ER stress on the mutation rate in our hypothesised hotspot of the exogenous human CSF3R, the corresponding region in the endogenous Csf3r, and Runx1. Ba/F3 cells transduced with the cDNA for partial C-terminal of CSF3R fused in-frame with a green fluorescent protein (GFP) tag were subjected to stress-inducing treatment for 30 days (~51 doubling times). The amplicon-based targeted deep sequencing data for days 15 and 30 samples show that although there was increased mutagenesis observed in all the three genes of interest (partial CSF3R, Csf3r and Runx1), there were more mutations in the GFP region compared with the partial CSF3R region. Our findings also indicate that there is no correlation between the stress-inducing chemical treatments and mutagenesis in Ba/F3 cells. Our data suggest that oxidative or ER stress induction does not promote genomic instability, affecting partial C-terminal of the transduced CSF3R, the endogenous Csf3R and the endogenous Runx1 in Ba/F3 cells that could account for these targets to being mutational hotspots. We conclude that other mechanisms to acquire mutations of CSF3R that help drive the evolution of SCN to MDS/AML.