Abstract
Introduction. Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency, and skeletal abnormalities. Patients with SDS are predisposed to develop myelodysplastic syndromes or acute myeloid leukemia. SDS is caused almost exclusively by biallelic mutations of SBDS. More recently, several cases of the SDS have been reported in those with germline EFL1 variants and wild-type SBDS. SBDS interacts with EFL1 to displace EIF6 from the pre-60S ribosomal large subunit and permits its assembly with the 40S ribosomal small subunit to form the 80S ribosome in the cytoplasm. SBDS deficiency leads to activation of p53 and CDKN1A (p21). Activation of p53 is thought to cause bone marrow failure. However, the pathophysiology of SDS is not fully understood, and mouse models have been of limited value. SBDS, EFL1, and EIF6 are highly conserved among eukaryotes. We reported sbds-null zebrafish showed neutropenia, decreased length, and pancreatic atrophy with upregulation of genes associated with cell cycle arrest, apoptosis, and lipid metabolism and accumulation of Eif6 protein. Mice with Efl1-missense mutations recapitulated several features of SDS such as weight loss, reduced bone mass density, and reduced bone marrow cellularity. Knockout of Efl1 resulted in embryonic lethality. Pancreatic exocrine insufficiency and skeletal abnormalities were not found in this murine model. Here, we report the phenotype of our efl1-null zebrafish.
Methods. We used CRISPR/Cas9 editing to generate a mutation in exon 7 of efl1 zebrafish, resulting in a premature termination codon that disrupts the GTP-binding domain. We bred efl1+/- fish and collected larvae at 5 and 15 days post fertilization (dpf).
Results. Genotype frequency of eggs fertilized from heterozygous carriers followed the expected Mendelian distribution. However, efl1-/- survival declined starting from 15 dpf. Standard length in efl1-/- was significantly reduced at 15 dpf, but not at 5 dpf. Neutrophils were significantly decreased in efl1-/- fish in comparison with those in efl1+/+ and efl1+/- siblings. The median number of neutrophils at 5 dpf was 36 (range, 17-79) in efl1-/-, whereas the median in efl1+/+ and efl1+/- was 93 (range, 29-193) and 84 (range, 17-151), respectively (P < .0001). At 5 dpf, RT-qPCR using whole larvae RNA showed significantly increased (4-8 fold) cdkn1a mRNA in efl1-/- fish compared to efl1+/+ and efl1+/- siblings. There was no significant upregulation of tp53 mRNA in efl1-/-. Accumulation of Eif6 protein in efl1-/- fish was found at 15 dpf, although eif6 mRNA were not significantly changed among efl1 genotypes. Because we reported in our sbds-null zebrafish aberrant fatty acid and cholesterol regulatory gene expression, we examined their expression in this efl1-null zebrafish. By 15 dpf, srebp1 mRNA was significantly higher in efl1-/-, and pparg and fasn also tended to be higher in efl1-/- in comparison with efl1+/+ and efl1+/-.
Conclusions. Zebrafish lacking efl1 phenocopied some of the molecular and morphologic features of SDS. Additionally, results from efl1-/- zebrafish were consistent with those from sbds-/- zebrafish strains, emphasizing a common molecular pathway induced by the dyad of EIF6 dissociating factors. Ongoing studies, which will be presented, are evaluating skeletal abnormalities and pancreatic atrophy in the efl1-/- zebrafish. Interestingly, sbds-null and efl1-null fish survived longer than eif6-null, suggesting more critical functions of EIF6. Altogether our data and presented elsewhere advance the hypothesis that CDKN1A and EIF6 contribute to the pathophysiology of SDS due to either mutations in SBDS or EFL1.
Disclosures
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
