Severe congenital neutropenia‐associated JAGN1 mutations unleash a calpain‐dependent cell death programme in myeloid cells

Abstract

Severe congenital neutropenia (SCN) of autosomal recessive inheritance, also known as Kostmann disease, is characterised by a lack of neutrophils and a propensity for life-threatening infections. Using whole-exome sequencing, we identified homozygous JAGN1 mutations (p.Gly14Ser and p.Glu21Asp) in three patients with Kostmann-like SCN, thus confirming the recent attribution of JAGN1 mutations to SCN. Using the human promyelocytic cell line HL-60 as a model, we found that overexpression of patient-derived JAGN1 mutants, but not silencing of JAGN1, augmented cell death in response to the pro-apoptotic stimuli, etoposide, staurosporine, and thapsigargin. Furthermore, cells expressing mutant JAGN1 were remarkably susceptible to agonists that normally trigger degranulation and succumbed to a calcium-dependent cell death programme. This mode of cell death was completely prevented by pharmacological inhibition of calpain but unaffected by caspase inhibition. In conclusion, our results confirmed the association between JAGN1 mutations and SCN and showed that SCN-associated JAGN1 mutations unleash a calcium-and calpain-dependent cell death in myeloid cells.

Kostmann disease and other forms of severe congenital neutropenia

“…Boztug et al 26 suggested as much, using peripheral blood neutrophils as a model, though the evidence for apoptosis was sparse and the underlying mechanism was not disclosed. We recently identified two different homozygous JAGN1 mutations in three patients with Kostmann disease, thus confirming the attribution of JAGN1 mutations to SCN 57 . We examined peripheral blood neutrophils from one of our patients (who responded poorly to recombinant G‐CSF treatment) and observed ultrastructural changes suggestive of aberrant granule exocytosis, but no signs of apoptosis (Figure 3).…”

“…Finally, we conducted a yeast two-hybrid screen and found that JAGN1 interacts with Grp78, a known regulator of the UPR as well as a regulator of calcium homeostasis in the ER. 57 In conclusion, our results showed that SCN associated JAGN1 mutations unleashed a calcium-and calpaindependent cell death in a human myeloid cell model. Further investigations are required to understand the role of calpain-mediated cell death in the pathogenesis of JAGN1-associated SCN.…”

“…We also noted that bongkrekic acid, a specific inhibitor of mitochondrial permeability transition pore opening, partially rescued the cells from calpain‐dependent cell death, pointing to a crosstalk between the ER and mitochondria. Finally, we conducted a yeast two‐hybrid screen and found that JAGN1 interacts with Grp78, a known regulator of the UPR as well as a regulator of calcium homeostasis in the ER 57 . In conclusion, our results showed that SCN associated JAGN1 mutations unleashed a calcium‐ and calpain‐dependent cell death in a human myeloid cell model.…”

“…To further study the impact of these JAGN1 mutations, we expressed both mutations in the human myeloid HL‐60 cell line, a commonly used model of myeloid cells. We found that overexpression of the patient‐derived JAGN1 mutations augmented cell death in response to classical pro‐apoptotic stimuli, including the chemotherapeutic agent, etoposide and thapsigargin, an inhibitor of the sarco/endoplasmic reticulum Ca 2+ ‐ATPase that triggers calcium‐dependent cell death 57 . Furthermore, we observed that cells expressing disease‐associated JAGN1 mutations were susceptible to agonists that trigger degranulation in neutrophils (i.e., the chemoattractant, fMLP, combined with the priming agent, cytochalasin D) and succumbed to a calcium‐dependent cell death programme 57 …”

“…We recently identified two different homozygous JAGN1 mutations in three patients with Kostmann disease, thus confirming the attribution of JAGN1 mutations to SCN. 57 We examined peripheral blood neutrophils from one of our patients (who responded poorly to recombinant G-CSF treatment) and observed ultrastructural changes suggestive of aberrant granule exocytosis, but no signs of apoptosis (Figure 3). To further study the impact of these JAGN1 mutations, we expressed both mutations in the human myeloid HL-60 cell line, a commonly used model of myeloid cells.…”