Gene Disruption of Zebrafish Sbds Phenocopies Human Shwachman-Diamond Syndrome but Suggests More Global and Lineage Defects

Introduction: Shwachman-Diamond Syndrome (SDS) is a complex disorder resulting in short stature, neutropenia, bony dysplasia, and pancreatic malabsorption. Individuals with SDS are also at increased risk for developing myelodysplastic syndrome and/or acute myeloid leukemia (AML). Its estimated incidence of 1 in 77,000 births makes SDS difficult to study and develop newer therapies. SDS results from mutations in the SBDS gene. Genetic ablation of Sbds results in early embryonic lethality (ED 7.5) in mice. Zebrafish provide an attractive, alternative model organism to study human diseases. Genome analysis of zebrafish (Danio rerio) revealed the presence of a single sbds gene, encoding a protein 90% identical to the human orthologue.

Methods: We used CRISPR/Cas9 genome editing technique to generate indel mutations in sbds andestablished multiple zebrafish lines. We used zebrafish sbds mutants to analyze the function of this gene during vertebrate development and in the pathophysiology of SDS.

Results: Western blotting showed progressive decrease of Sbds expression with no detectable protein at 8 days post fertilization (dpf). These results suggest that the presence of Sbds during early stages of the development is due to maternal deposition, which was confirmed by qPCR and immunoblot demonstration of sbds transcript and proteins in in all stages of the development including the 1 cell-stage. Unlike Sbds-/- mice, homozygous mutant sbds^nu132live up to 6 weeks. They display marked growth retardation. We also observed a significantly low number of neutrophils at 5 dpf and 15 dpf. No differences were found in macrophages and hemoglobinization. Interestingly, multiple organs, especially the pancreas, liver, digestive tract and eye showed histologic evidence of atrophy at 21 dpf. Differences were also observed in fat deposition and bone formation. These defects were not seen at 15 dpf, suggesting a critical stage in development dependent on functional sbds.

Conclusions: To our knowledge, we established the first zebrafish model of SDS, making it a highly relevant model to investigate its pathophysiology and develop new small molecule therapies. Comparison of null states between mice and zebrafish suggest that loss of Sbds results in two phenotypes: an early embryonic stage required for viability and development and a post-embryonic (larval) stage critical for granulocyte production and normal size. Data will be presented on bone formation, neutrophil chemotaxis, metabolomics, and RNAseq in the zebrafish model.

This work was supported by grants from the NIH R21CA15920, Shwachman-Diamond Syndrome Foundation and the Alex Turnquist Memorial Research fund of Shwachman-Diamond America.