Mutations in the ELA2 Gene Encoding Neutrophil Elastase Induce the Unfolded Protein Response and May Contribute to Neutropenia through the UPR-Dependent Apoptosis of Granulocytic Precursors.
Authors: Grenda David S. , Link Daniel C. 
Published: April 2020

Abstract
Severe congenital neutropenia (SCN) is a group of disorders characterized by severe neutropenia, an accumulation of promyelocytes in the bone marrow, and a predisposition to myelodysplasia (MDS) and acute myeloid leukemia (AML). While SCN is genetically heterogeneous, linkage analysis has shown a strong correlation between mutations in the ELA2 gene encoding neutrophil elastase (NE) and the development of SCN. These mutations are all heterozygous germline mutations, suggesting a gain-of-function mechanism of action. However, a common biochemical mechanism by which these mutations contribute to neutropenia has not yet been elucidated. Specifically, the mutations identified to date have no consistent effect on protease activity or on the subcellular localization of NE. We therefore investigated the possibility that these mutations disrupt the structure of NE, leading to the induction of the unfolded protein response (UPR). The UPR is a coordinated program of compensatory gene expression and regulation which protects the cell from the accumulation of misfolded proteins. When the quantity of misfolded protein overwhelms the compensatory mechanisms initiated by the UPR, the cell will undergo apoptosis.
To test the hypothesis that mutations in ELA2 lead to the production of misfolded NE and induction of the UPR, we employed a transient transfection assay in K562 cells. Specifically, cells were transfected with a vector containing wild type or V72M, P110L, G185R, R191Q, or G192pter mutant NE linked to GFP by an IRES motif. To assess the role of NE protease activity, protease deficient double mutants of V72M and P110L were generated by replacing the catalytic serine (S173) with alanine. At 12 hours post transfection, cells were sorted on the basis of GFP expression and induction of the UPR assessed by two assays: induction of the ER chaperone Grp78/BiP and splicing of XBP-1 mRNA. Compared with WT NE, expression of the different NE mutants resulted in a 2–6 fold increase in BiP mRNA expression. Moreover, the ratio of spliced to unspliced XBP-1 mRNA increased 2–10 fold in cells expressing mutant versus WT NE. Intriguingly, the degree of UPR induction correlated with the severity of the phenotype observed in patients with the various mutations analyzed in our study. For example, a 8-fold induction in BiP mRNA was detected in cells expressing the SCN-associated V72M mutant whereas only a 2-fold induction was detected in R191Q NE expressing cells.
To verify that UPR induction correlates with loss of cell viability, single cells were plated 12 hours after transfection in 96 well plates and clonogenic capability was determined by scoring positive wells at 2 weeks post sorting. In preliminary experiments, the impairment in clonogenic potential correlated with the degree of UPR induction, suggesting a link between NE expression, UPR induction, and cell death. Of note, the protease-deficient double mutants were as effective as the single NE mutants in inducing the UPR and impairing clonogenic potential.
Collectively, these data suggest that the expression of mutant NE is sufficient to induce the unfolded protein response and induce cell death in K562 cells. Studies are currently underway to verify these findings in human promyelocytes. These data provide a clear and consistent mechanism for the apoptosis of granulocytic precursors seen in SCN. More importantly, this would be the first known case of a congenital disorder caused by apoptosis secondary to misfolded proteins.