SRP54 mutations induce Congenital Neutropenia via dominant-negative effects on XBP1 splicing

Heterozygous de novo missense variants of SRP54 were recently identified in patients presenting with Congenital Neutropenia (CN) or its syndromic form Shwachman-Diamond Syndrome (SDS). Ever since its discovery as a driver of CN and SDS, SRP54 has been increasingly studied in the context of disease and is nowadays considered the second most common cause of CN. Despite its hitherto unknown prevalence, the molecular mechanisms leading to the development of the disease are still largely unknown and patient treatments are far from specific.
In this thesis, I aimed to investigate the underlying mechanisms and processes contributing to the pathophysiology of SRP54 deficiencies. To follow this aim, I characterized and established a transgenic srp54 KO zebrafish as the first in vivo model of srp54-driven disease. Interestingly, srp54-/- zebrafish show early embryonic mortality and suffer from severe neutropenia and developmental defects affecting multiple organs. srp54+/- zebrafish on the other hand are viable and only display mild neutropenia and no overt other defects. However, when injecting srp54+/- fish with human mRNA of three mutated SRP54 variants (T115A, T117Δ and G226E) identified in patients, the neutropenia intensified, and pancreatic defects developed – a phenotype accurately mimicking the characteristics of SDS patients. Of note, the induced phenotypes showed mutation-specific differences, indicating that different SRP54 lesions exert unique dominant-negative effects on the functionality of the residual wildtype SRP54 protein. Consistent with these findings, overexpression of SRP54 missense variants in human promyelocytic HL60 cells as well as in healthy CD34+ cord blood cells impaired granulocytic maturation.
Mechanistically, we found that SRP54 defects significantly reduce the efficiency of the unconventional splicing of the transcription factor X-box binding protein 1 (XBP1), which is one of the major regulators of the unfolded protein response (UPR). Vice-versa, xbp1 morphant zebrafish recapitulate phenotypes observed in srp54 mutant fish, and the injection of spliced xbp1 but not unspliced xbp1 rescues the neutropenia in srp54+/- embryos.
In order to identify additional mechanisms contributing to the pathophysiology of SRP54 deficient patients, we performed single cell RNA sequencing of srp54-mutated zebrafish. Sequencing analysis revealed several differentially expressed genes with most of them converging on the major signaling branches of the UPR, indicating the cell’s efforts to circumvent the impaired XBP1 activity aiming to alleviate unresolved ER-stress.