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Page 6 of 10 Mazur et al. Rare Dis Orphan Drugs J 2023;2:1 https://dx.doi.org/10.20517/rdodj.2022.12

decreased expression of genes encoding key hematopoietic transcription factors as a result of the production
of pathogenic NE variants [Figure 1C].

The most prevalent theory of how altered NE produces a neutrophil deficit is that mutations in ELANE
result in improper folding, triggering the stress response pathway (UPR) within the ER, which leads to
apoptosis of neutrophil precursors. The expression of incorrectly folded NE variants can be expected to
culminate in promielocytes stage of granulopoiesis, when NE becomes the most abundant protein. The bulk
of misfolded NE is, therefore, likely to accumulate at high levels in the ER and induce the UPR response
followed by cell death. This mechanism is supported by experimental evidence, showing typical markers of
the canonical UPR pathways, such as activation of molecular sensors (ATF6, CHOP) and master regulator
of ER stress (GRP78), in cell models expressing pathogenic NE mutants [24,25] .

However, not all neutropenia-causative NE variants are capable of consistently evoking the classic UPR
response in experimental cell models. Degradation of misfolded proteins, independent of the canonical
[26]
UPR, can also be a consequence of other protective cell strategies . Recent findings point to elevated levels
of promyelocyte leukemia protein nuclear bodies (PML-NBs), an indicator of acute oxidative stress, which
may provide an alternative protective strategy in cells that harbor misfolding NE mutations. PML-NBs can
be formed to diminish excessive ROS levels in the affected cells, but the presence of PML-NBs in cells with
NE misfolding variants also stimulates cell metabolism and boosts the expression of NE, including NE
[26]
mutants . Thus, the formation of PML-NBs, independent of canonical UPR responses, may further
promote neutropenic state.

Many NE mutations are predicted not to lead to NE misfolding, since they do not substantially interfere
with the structure of the core protein . Instead, they may be responsible for the failure of NE to be
[26]
properly distributed within the cells. The first clue regarding how mutant NE can contribute to neutropenia
and support for mislocalization theory came from studies in dogs . Dogs suffer from CyN, which is similar
[1]
to the human version, but canine CyN is autosomal recessive and does not result from NE but from AP3
mutation. AP3 is a protein involved in the trafficking of cargo proteins from the trans-Golgi network to
lysosomes, suggesting that AP3 may play a role in NE trafficking and accumulation in granules. If NE, in
order to be distributed in granules, interacts with AP3, a mutation in either gene could cause disruption to
the intracellular trafficking of NE following the accumulation of NE in inappropriate subcellular
compartments. An altered NE distribution in AP3-deficient dogs with canine cyclic neutropenia is in line
with NE, which serves as an AP3 cargo protein . Therefore, chain-terminating C-terminal NE mutations
[1]
that disrupt NE interaction with AP3 could be expected to be mislocalized within the cells. This is what is
observed in SCN patients .
[1]
Among other mistrafficking, NE mutations are, for example, NE variants that lack the ER localizing signal
sequence, which is associated with transcriptional start site mutations . The mistrafficking of NE and its
[27]
accumulation in the cytoplasm beneath the plasma membrane or in cytosol has been reported for several
NE variants [28-30] . Furthermore, cells that express the pathogenic G185R NE mutant associated with clinically
severe forms of neutropenia, or that express shorter NE isoforms, exhibit a different distribution pattern of
NE with localization to the nuclear and plasma membrane or nuclei [27,31] . Either proteolytically active or
inactive NE mutants appear to be associated with off-site intracellular accumulation [22,28,30] . However, the
proteolytic activity of these mislocalized NE variants has yet to be imaged in intact cells. The atypical
intracellular distribution of NE might result in increased immunoreactivity for NE, a lack of control by
[16]
intracellular inhibitors, and a lower activation threshold in neutrophils . Taken together, these data suggest
that the mislocalization of NE either via mutation of NE itself or in NE adaptive proteins, such as AP3