Mutation-Specific Dominant Negative Effects Determine the Phenotype in SRP54 Deficiency
2019-11-13

Whole exome sequencing analyses are increasingly performed on patients presenting with suspected inherited disease but lacking classical mutations linked to presented phenotypes. Using whole-exome sequencing in SBDS-negative Shwachman-Diamond Syndrome (SDS) families, we recently identified three independent patients, each of whom carried a heterozygous de novo missense variant of SRP54 (encoding signal recognition particle 54 kDa). The SRP54 protein is a key component of the ribonucleoprotein complex that mediates the co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum (ER). Whilst two of the identified patients were carrying nucleotide transversion in SRP54 (p.T115A and p.G226E), which manifested in typical SDS features like neutropenia and exocrine pancreatic insufficiency, the third patient was carrying a nucleotide deletion (p.T117Δ), which only manifested in mild neutropenia without additional SDS features (Carapito et al. 2017, JCI). Here, we describe a zebrafish knock-out (KO) mutant as the very first transgenic in vivo model of SRP54 deficiency, translate our previous findings into living organisms and propose disease-driving mechanisms. We show that homozygous srp54 mutant zebrafish are suffering not only from severe neutropenia as shown by flow cytometry and Whole-Mount-In-Situ Hybridization (WISH), but also from gross developmental defects leading to early embryonic lethality. In fact, srp54-/- zebrafish did not survive more than 72 hours post fertilization, indicating that complete loss of Srp54 is not compatible with life. Injection with wild-type human SRP54 mRNA induced transient restoration of SRP54 protein expression and slightly enhanced the survival of the homozygous mutants. However, long-term viability could not be restored, revealing that srp54 is not only critically required during early embryogenesis but also at later stages of development. Heterozygous siblings on the other hand are viable and display only mild neutropenia but no pancreas defects. Interestingly however, injection of mutant mRNAs of human SRP54 (p.T115A, p.T117Δ, p.226E) into heterozygous srp54 KO mutants aggravated the phenotype inducing more profound neutropenia and pancreas changes similar to those observed in classical SDS patients. Of note, these effects were more severe for the transversions p.T115A and p.G226E. Mutation p.T117Δ only caused a minor reduction in the number of neutrophils, without affecting the pancreas. To further investigate SRP54 driven neutrophil defects, we used lentiviral transduction to exogenously express human SRP54 mutant variants in promyelocytic HL-60 cells. When stimulating these cells to differentiate by ATRA treatment, we found significantly impaired morphologic differentiation and CD11b surface induction compared to control cells. The severity of these effects was again specific to the three different identified mutations, with p.T115A and p.G226E being more severe than p.T117Δ. These findings confirm the type-specific effects of SRP54 mutations and indicate that SRP54 defects interfere with neutrophil differentiation and thus ultimately lead to neutropenia. Collectively, we here describe a novel zebrafish disease model of SDS and congenital neutropenia founding on SRP54 as molecular driver. Our model demonstrates that at least one healthy allele of srp54 is pivotal for survival, which is in line with the findings in humans, where homozygous mutations in SRP54 have never been detected. We reveal that the phenotypic manifestation of heterozygous SRP54 mutations strongly depends on the type of mutation: while mutations likely causing a simple SRP54 loss of function (e.g. p.T117Δ) induce a rather mild phenotype characterized by moderate neutropenia only (analogous to the heterozygous fish mutant), more severe SDS-like phenotypes involve SRP54 mutations that exert dominant negative effects (e.g. p.T115A and p.G226E). Ultimately, we make use of the promyelocytic cell line HL-60 to propose neutrophil differentiation defects as the underlying cause of SRP54 driven neutropenia. At the time being, RNA sequencing and protein expression analyses are performed in our laboratory, which will add to the understanding of the mechanistical background of the neutrophilic differentiation blockage and eventually uncover novel treatment strategies for SRP54 deficiency. Disclosures No relevant conflicts of interest to declare.