Zebrafish Study Identifies Genetic Rescue Mechanism in Shwachman-Diamond Syndrome

Using a novel zebrafish model, Cleveland Clinic investigators have identified a genetic mechanism in the TP53 pathway that improves the course of Shwachman-Diamond syndrome (SDS). The breakthrough, presented in a poster atthe 66^th American Society of Hematology Annual Meeting, may point the way toward development of novel drug therapies for patients with this rare pediatric inherited bone marrow failure syndrome.

“Our gene expression analysis in the world’s only zebrafish model of Shwachman-Diamond syndrome identified a cellular stress response involving TP53,” says Seth J. Corey, MD, a pediatric oncologist at Cleveland Clinic Children’s and a professor in Pediatrics and Molecular Medicine at Cleveland Clinic, who led the study. “The results suggest that low levels of the EIF6 gene can mitigate that pathway, partially alleviating cellular stress in cells deficient in the SBDS gene, which are critical to development of the syndrome and increases the risk of leukemia.”

Understanding Shwachman-Diamond syndrome

An autosomal recessive disorder, SDS is rare, occurring in 1 in 75,000 to 1 in 120,000 live births. Exocrine pancreatic insufficiency, neutropenia and skeletal abnormalities are the hallmarks of the condition, which also is associated with an increased risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).

In children, 90% percent of all cases of SDS are caused by biallelic mutations in the SBDS gene, which is critical to ribosome assembly. SBDS interacts with the ribosomal protein EFL1 to remove the Eif6 protein from the 60S ribosomal subunit, facilitating ribosome formation in the cytoplasm. Ribosomes are complex cellular “machines” that produce proteins from messenger RNA.

Previous research on mouse and zebrafish models implicated SBDS deficiency in SDS and P53 mutations have been found in some patients with MDS/AML. However, prior to the new study, the mechanism of somatic genetic rescue in SDS had not been characterized.

Innovative model and investigation

Created in Dr. Corey’s lab with CRISPR/Cas9 editing, the zebrafish model of SDS is a close phenocopy of the human condition, deficient in the genes that cause SDS. Zebrafish were chosen for the research because of their distinct advantages over mouse models: organogenesis within 96 hours, transparent development, prodigious production of eggs that afford statistical significance and low costs of maintenance and breeding.

“Fortuitously, the similarity between human and zebrafish SDS proteins is almost 90%,” says Dr. Corey. “The zebrafish model has characteristics of children with SDS in that they have small stature, neutropenia and pancreatic atrophy.”

For the latest research, the investigators created SBDS-deficient zebrafish that exhibited Eif6 accumulation, changes in levels of ribosome proteins and activation of TP53 pathways. An Eif6 knockout line was created, which died earlier than the line without the knockout (7 to 10 days vs. 15 days post-fertilization).

“To determine how protein levels of Eif6 affected SDS, we generated zebrafish mutants with low Eif6 protein expression. Surprisingly, we observed that these Eif6 hypomorph mutants survived to adulthood,” says Dr. Corey. “Polysome profiling revealed significant reductions in the 80S monosomes and 40S ribosomal subunits in the Eif6 knockout at 5 dpf. However, the EIF6 hypomorph mutants displayed polysome profiles similar to the EIF6 wild type.”

Next, the EIF6 knockout or hypomorph mutants were crossed with the SBDS knockout fish and their phenotypic and molecular characteristics were analyzed. In SBDS-deficient fish, lower levels of EIF6 significantly increased survival. In the SBDS­-deficient fish, however, low EIF6 levels did not rescue neutropenia.

Notes Dr. Corey, “Expression of TP53-dependent targets was reduced in the Eif6 hypomorph mutants with an SBDS-null background. SDS patient-derived cell lines also showed accumulation of EIF6, P53 and CDKN1A, and knocking down Eif6 significantly decreased CDKN1A mRNA levels.”

Future research and clinical implications

The findings, according to the researchers, underscore the complex relationships between SBDS, Eif6, TP53, and stress responses in the pathogenesis of SDS. Eif6 dosage, they believe, determines the degree of intracellular stress mediated by TP53, which explains the somatic genetic rescue in SDS and may decrease the risk of transformation to leukemia.

“Our results indicate that there are genetic mutations that can help rescue fish from the deleterious effects of the SBDS mutation and that modulating those mutations up or down can improve survival and appearance,” says Dr. Corey. “The data are being validated and the next step is to take this information and attempt to develop drugs that mimic the somatic genetic rescue and help prevent leukemia in patients with SDS.”