Background. Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic insufficiency, skeletal defects, neutropenia, and an increased risk of myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). SDS occurs in 1/75,000 births, and biallelic mutations in the SBDS gene account for ~90% of patients. The SBDS protein is highly conserved. SBDS interacts physically with EFL1 to release EIF6 from the cytoplasmic pre-60S ribosomal subunit and promote the assembly of the mature 80S ribosome. The SBDS R126T allele is found in combination with the common K62X mutation in some SDS patient. A recent study showed that the SBDSR126T is not able to activate the GTPase activity of the EFL1, affecting the release of EIF6 from the 60S surface.

Methods. We created a zebrafish knockout line that phenocopies the SDS with neutropenia, pancreas atrophy, small size (Figure 1A), and decreased 80S ribosomes. To rescue those fish from early mortality, we generated a new transgenic line Tg(ubi:SBDSR126T:pA) expressing the missense variant R126T, a disease-associated allele.

Results. The sbds knockout fish die after 21 days post fertilization (dpf), corresponding to an early juvenile stage. However, the SBDSR126T transgenic line in the background of the sbds knockout can live for at least 12 months. This is in strong contrast to the mouse SbdsR126T/R126T line that do not survive to birth. Transgenically-rescued fish displayed a small size phenotype resembling SDS (Figure 1B). Levels of ribosomal proteins Rpl5 and Rpl11 were lower in the sbds knockout at 21 dpf but they were normal in the transgenic line at 6 months. We also observed a concordant regulation of Sbds and Eif6 expression (Figure 1C,D). sbds null fish showed a significant upregulation of cdkn1a, while in their transgenic siblings levels were normal (Figure 1E). Moreover, mpx was upregulated in the transgenic line with the null background (Figure 1F). Analysis of neutrophil and monocyte counts are being performed and will be reported.

Conclusions. Our novel SBDSR126T zebrafish model survives until adulthood, which will allow us to carry out a number of informative assays such as stress response, gene expression, and polysome profiles in different organs. Rpl5 and Rpl11 levels are affected in sbds mutants but not in the transgenic line. Activation of cdkn1a (p21) in sbds mutants might lead to apoptosis and death. The normal levels of cdkn1a in the transgenic line might be non-deleterious, as loss of Tp53 activation can rescue some models of bone marrow failure. In addition, loss of sbds or expression of SBDSR126T affect Eif6 levels in zebrafish. Importantly, some patients with SBDS deficiency acquire interstitial deletions of chromosome 20, resulting in the loss of the EIF6 gene. This might be a potential mechanism to suppress the defect in ribosome biogenesis by reducing the copy number of the EIF6 gene and has been related to a lower risk of MDS/AML comparing to other SDS patients. Our adult model of Shwachman-Diamond Syndrome can provide new insights into the pathogenesis of SDS and its progression to malignancy, which can be used to identify novel targets for AML/MDS therapy.

Figure 1
Figure 1
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Disclosures

No relevant conflicts of interest to declare.

Topics:
bodily distress syndrome, shwachman syndrome, zebrafish, neutropenia, antimicrobial chemoprophylaxis, atrophy, autosome disorder, cancer, death, guanosine triphosphate phosphohydrolases

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2019 by The American Society of Hematology
2019
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