Mutation-Specific Dose Reduction in Functional SRP54 Protein Causes Isolated or Syndromic Neutropenia with Shwachman-Diamond-like Features

Sequencing analyses are increasingly performed on patients presenting with suspected congenital disease but lacking classical mutations linked to the presented phenotype. However, in case a novel mutation is found, its causal contribution to the patients' clinical symptoms is yet unclear and requires further exploration in functional studies. Here we use the zebrafish model to analyze the functional relevance of heterozygous SRP54 gene mutations identified in patients with unexplained neutropenia (T117del) or Shwachman-Diamond like disease involving severe neutropenia and exocrine pancreas insufficiency (T115A, G226E) (Carapito et al, 2017). The function of wildtype SRP54 protein was explored in hematopoiesis and pancreas development using two different antisense morpholino oligonucleotides (MO) and a zebrafish srp54 mutant. Reduced neutrophil numbers were observed in MO versus control injected fish when analyzed by WISH for mpx or using Tg(lyz:DsRed) and Tg(mpx:eGFP) lines. Morphants displayed not only quantitatively but also qualitatively impaired neutrophils, which migrated less to injury sites in tail fin injury assays. Furthermore, exocrine but not endocrine pancreas impairment was observed in MO versus control injected fish. Both neutrophiles and exocrine pancreas development could be rescued by co-injection with wildtype SRP54 mRNA.

Interestingly, in the corresponding Sanger mutant, homozygous srp54^-/- fish display not only most severe neutropenia but also gross developmental defects which cause early lethality at 2 days post-fertilization and thus impede assessment of pancreas development. Heterozygous srp54^+/- fish are viable, show less severe neutropenia than homozygous fish and, interstingly, have no exocrine pancreas defect. The delineated experiments explore thus the effects of different dosages of residual functional SRP54 protein (wildtype > srp54^+/- > morpholino treated > srp54^-/-) on phenotypic manifestation. The obtained results indicate that the dose of residual SRP54 protein that can functionally act within the SRP may dictate disease phenotype in patients with SRP54 mutations (neutropenia only > more severe neutropenia and exocrine pancreas defects > most severe neutropenia, early lethality).

We thus hypothesize that the different mutations observed in patients differentially affect the functionality of the residual wildtype SRP54 protein and thus associate with isolated neutropenia (T117del) or more severe disease with Shwachman-Diamond like features (T115A, G226E). To mimic the heterozygote mutation status observed in patients, we injected the different mutated mRNAs in srp54^+/- zebrafish. None of the injected mRNAs was able to rescue the neutropenia phenotype, indicating that all mutated proteins are functionally impaired. Confirming our hypothesis, injection of T115A and G226E in fact further aggravated neutropenia and additionally induced exocrine pancreas defects, while T117del did not.

Taken together, these analyses support the notion that full deletion of SRP54 is not compatible with life and therefore homozygous SRP54 are not observed in patients. Instead, heterozygous SRP54 mutations may cause only neutropenia or alternatively the more severe Shwachman-Diamond like syndrom, depending on the nature of the underlying mutation (either functional null or dominant negative).