Mutations of the ELA2 Gene Found in Patients with Severe Congenital Neutropenia Induce the Unfolded Protein Response and Cellular Apoptosis.

Severe congenital neutropenia (SCN) is characterized by severe chronic neutropenia and promyelocyte accumulation in the bone marrow. Mutations in the ELA2 gene encoding neutrophil elastase (NE) are responsible for most cases of SCN and nearly all cases of cyclic neutropenia (CN), a related but milder disorder of granulopoiesis. To date, 47 distinct, mostly missense, mutations have been reported in patients with CN or SCN; most segregate with one phenotype, suggesting a genotype-phenotype correlation. While genetic studies suggest that ELA2 mutations act in a dominant, cell-intrinsic fashion to disrupt granulopoiesis, the molecular mechanisms by which they do so are unknown. Given the functional heterogeneity of NE mutants in SCN, we hypothesized that ELA2 mutations disrupt granulopoiesis by leading to the production of NE proteins that misfold, activate the unfolded protein response (UPR), and ultimately trigger apoptosis in granulocytic precursors. The UPR is a well-characterized cellular program that acts to ameliorate the accumulation of misfolded proteins in the endoplasmic reticulum (ER) via general attenuation of translation initiation, upregulated expression of ER resident protein chaperones, and increased ER-associated degradation (ERAD) of misfolded proteins. Persistence of the folding defect promotes apoptosis.

We previously showed that expression of mutant NE induces BiP/GRP78 gene expression, a classic biochemical marker of the UPR, and impairs clonogenic capacity in a myelomonocytic cell line. To further explore the UPR hypothesis, we employed a transient transfection assay in which granulocytic precursors cultured from human cord blood-derived CD34⁺ cells express wild type or mutant forms of NE associated with SCN (V72M, G185R, G192pter), CN (R191Q), or both SCN and CN (P110L). Here we show that:

1. Expression of SCN-related NE mutants but not R191Q NE induces BiP mRNA expression and XBP1 mRNA splicing, classic markers of the UPR;

2. The degree of the UPR induced by each mutant in our study roughly correlates with the severity of its associated clinical phenotype. Notably, G185R NE, associated with the most severe clinical phenotype, induces the greatest BiP expression;

3. Expression of SCN-related NE mutants is associated with increased apoptosis; and

4. Protease-deficient double mutant forms of NE still induce the UPR and trigger apoptosis, suggesting that mutant NE disrupts granulopoiesis by a protease-independent mechanism.

We next analyzed primary granulocytic precursors from 6 ELA2-positive SCN patients and 5 healthy donors and detected a 5.7-fold increase in BiP mRNA expression (p=.06) and a 2.5-fold increase in XBP1 mRNA splicing (p=.03) in the SCN samples. In addition, confocal microscopy of normal and ELA2-positive SCN bone marrow cells stained for NE reveals a marked reduction in NE expression in SCN cells, consistent with the UPR hypothesis. Together, these data strongly support a UPR model of SCN disease pathogenesis, placing SCN in a growing list of human diseases caused by misfolded proteins. More importantly, SCN represents the first known case of a congenital disorder caused by UPR-induced apoptosis.