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Background: We developed a WES-based pipeline for finding new severe congenital neutropenia (CN) causative gene variants for patients without known underlying genetic defects. We have identified a novel homozygous stop-codon mutation p.Gln141Ter in the COPZ1 gene in two siblings with CN, signs of primary immunodeficiency and mental retardation. COPZ1 encodes a subunit of the coatomer protein complex I (COPI), which is involved in intracellular protein trafficking and autophagy. While COPZ1 is ubiquitously expressed, its paralogue, COPZ2, is not detected in blood and brain tissues.
Aims: To investigate the role of COPZ1 mutation in defective granulopoiesis in vitro and in vivo.
Methods: We introduced COPZ1 mutation in cord blood (CB) hematopoietic stem and progenitor cells (HSPCs) of healthy donors, induced pluripotent stem cells (iPSCs), AML cell line NB4 and zebrafish embryos using CRISPR/Cas9 gene-editing. We performed in-silico simulations of the mutant COPZ1 protein using the atomistic molecular dynamics program CHARMM. In addition, we conducted in vitro liquid culture differentiation (LCD) and colony-forming units (CFU) assays of COPZ1 mutant CB-HSPCs and embryoid body-based granulocytic differentiation and Western Blot of COPZ1 mutant iPSCs. Furthermore, we treated COPZ1 mutant NB4 with Tunicamycin to induce unfolded protein response (UPR) stress. Finally, we performed RNA-sequencing of COPZ1-mutated and control-edited HSPCs.
Results: WB showed that COPZ1 mutation p.Gln141Ter results in the expression of truncated COPZ1 protein. CHARMM predicted that truncated COPZ1 alone and within the COPI complex displays significant structural instability compared to the full-length COPZ1, arguing for a disturbed COPI complex formation in the presence of truncated COPZ1. Compared to control edited cells, COPZ1-mutant CB-HSPCs have abrogated granulopoiesis in LCD and CFU assay. In vitro granulocytic differentiation of COPZ1-mutant iPSCs was also severely affected. In addition, copz1 mutant zebrafish embryos had a markedly lower number of neutrophils than the wild-type group. Furthermore, we confirmed that transduction of COPZ1 mutated HSPCs with WT COPZ2 rescued defective granulopoiesis triggered by truncated COPZ1. Thus, the organ-specific expression of COPZ2 may explain the neuro-hematological symptomatology and provide therapeutic opportunities.
Analyses of RNA-seq data from mutant vs WT cells revealed a significant up-regulation of the NFKB and STING pathways and down-regulation of the JAK/STAT signalling pathway in mutant cells. STING expression is associated with inflammation and endoplasmic reticulum (ER) stress. Compared to the control group, COPZ1-mutant NB4 cells showed higher sensitivity to the tunicamycin-induced UPR as ER stress. Thus, we propose that COPZ1-mutant cells accumulate STING in the Golgi due to ineffective ER retrieval, triggering inflammation via the NFKB pathway and UPR/ER stress. Intriguingly, COPZ1-mutant HSPCs showed downregulation of CLU, SRC, BNIP3/BNIP3L, and upregulation of TIGAR and NPC1 genes, all signs of suppressed autophagy.
Summary/Conclusion: This is the first report on an immunodeficiency syndrome with CN caused by a homozygous stop-codon mutation in the COPZ1 gene. As a potential pathomechanism for neutropenia downstream of COPZ1-mutation, we suggest a pathological positive feedback between the STING inflammatory pathway and insufficient autophagy associated with UPR/ER stress. Finally, the rescue of COPZ1 mutated HSPCs with COPZ2 overexpression corrected the defective granulopoiesis, suggesting promising therapeutic avenues.
