Studies of granulocyte colony stimulating factor signaling to develop tools for clinical assessments of severe congenital neutropenia

Abstract

Neutropenia is condition characterized by low number of neutrophils in circulation. This leads to an increased risk of infections and is often diagnosed early in life. Extreme cases are known as severe neutropenia and are often associated with inactivating mutations in common neutrophil genes. Granulocyte colony stimulating factor receptor (G-CSFR), encoded by CSF3R, is a growth factor receptor known to induce stem cell release from bone marrow (BM), but also stimulate granulopoiesis. Mutations in this gene leading to low neutrophil counts are associated with the disease severe congenital neutropenia 7 (SNC7). Classification of genetic variants or mutations based solely on sequencing data can be subjective and often lead to misclassification. Pathogenic mutations have the potential to be categorized as variants of uncertain significance (VUS). This also includes mutations in CSF3R, where missense mutations can have a large impact on neutrophile production. A functional test to assess the effects of novel missense mutations in the CSF3R gene would be beneficial for the diagnostic work up of SCN7 patients. This project aims to establish cellular assays for the investigation of G-CSF signaling and for functional characterization of CSF3R mutations. First, an assay was created using phospho-flow cytometry to study changes in protein signaling after G-CSF stimulation of primary human blood cells. Secondly, a reporter assay was developed for assessing the impact of CSF3R-mutations on STAT3 signaling. A specific mutation previously classified as a VUS, p.(Gly27Arg) was also studied to characterize its impacts on STAT3 signaling following receptor stimulation. Stimulation assays on neutrophils showed a lack of signaling downstream from the G-CSF receptor. We did, however, observe significant signal transduction in the phospho-flow assay for two out of the four signaling proteins (STAT3 and STAT5) when studying monocytes. The reporter-assay was successful in quantifying STAT3 signal after G-CSF stimulation but showed some difficulties with activating mutations. The mutation p.(Gly27Arg) was found to be likely pathogenic with STAT3 signaling barely detectable compared to WT. Both assays created show promising results for clinically validating the significance of CSF3R variants.