Impact of novel genetic variants in HYOU1 on neutrophil phenotype Free

Neutrophils, the most abundant leukocytes in the innate immune system, are crucial for defending against pathogens at the frontline. Severe congenital neutropenia (SCN) is a hereditary hematologic disorder marked by severe, chronic neutropenia and recurrent bacterial infections starting in early infancy. To date, around 20 causative genes for SCN have been identified, including ELANE, HAX1, G6PC3, GFI1, JAGN1, and SRP54. Many of these genes are linked to the endoplasmic reticulum (ER), which plays pivotal roles in protein synthesis, post-translational modifications, protein folding, and protein homeostasis. Hypoxia up-regulated 1 (HYOU1), a stress-induced chaperone located in the ER lumen, is also known as a responsible gene for SCN. Its deficiency contributes to neutropenia, as well as defects in B cells and dendritic cells. As a member of the heat shock protein 70 family, HYOU1 serves as a chaperone molecule, activated by external stressors such as ER stress, hypoxia, ischemia, and hypoglycemia.

Here, we identified novel homozygous variants in HYOU1 in two unrelated patients: a 10-year-old girl (patient 1, P1) and a 3-month-old boy (patient 2, P2), both presenting as small for gestational age and exhibited failure to thrive, along with neutropenia, hypogammaglobulinemia, and B-cell deficiency. Additionally, P1 displayed abnormal glucose metabolism, mild mental retardation, and dysmorphic facial features. The variants were p.L469P (P1) and p.F179V (P2), respectively. The transferrin isoform profile of P2 showed increased disialo- and trisialo-transferrin with decreased tetrasialo-transferrin, indicating N-glycosylation defects. Immunophenotyping of peripheral blood mononuclear cells (PBMCs) from both patients confirmed severe B-cell loss, with the remaining B cells being transitional or naïve, and no memory B cells detected. Additionally, the total percentage of NK cells was reduced in both patients. P1 started IVIG at 2 years of age, and P2 started IVIG at 1 month of age and G-CSF at 3 months of age.

To functionally validate these novel genetic variants, we introduced patient-specific variants (HYOU1 p.L469P and p.F179V, and a previously reported variant p.Y231H) into healthy donor-derived iPS cells (iPSCs) using CRISPR-Cas9 editing and differentiated these engineered cells into neutrophils. Unlike HD-iPSCs, HYOU1-mutated iPSCs showed reduced neutrophil differentiation, increased Caspase 3/7 activity, and impaired maturation. Colony assays using the CD34-positive cells also demonstrated a decrease in granulocyte colonies in HYOU1-mutated iPSCs as opposed to HD-iPSCs, suggesting that HYOU1-mutated iPSCs had a reduced capacity to differentiate into neutrophil compared to HD-iPSCs. We also corrected the p.L469P variant in patient-derived iPSCs (PD-iPSCs) and differentiated them into neutrophils. The corrected iPSCs exhibited higher viable cell counts, reduced Caspase 3/7 activity, and restored maturation compared to uncorrected PD-iPSCs. These findings confirm that the novel variants in HYOU1 are responsible for the phenotype of neutropenia observed in the patients.

To investigate the effect of HYOU1 variants at the protein level, a comparative proteomic analysis of peripheral blood neutrophils was conducted using samples from patients with variants in HYOU1, as well as in ELANE and SRP54, alongside samples from a healthy donor (HD). The UMAP analysis clearly distinguished between normal and SCN clusters. Within the SCN clusters, the proteomic profile of P1 formed a distinct cluster, separate from the proteomes of patients with variants in ELANE and SRP54. Gene ontology analysis revealed that HYOU1-mutant neutrophils specifically exhibited a reduction in proteins related to Coat Protein Complex II (COPII) vesicle coating, ER-to-Golgi vesicle-mediated transport, and vesicle targeting compared to those from HD or SCN patients with ELANE or SRP54variants. This suggests HYOU1 may be involved in intracellular trafficking from the ER to the Golgi in neutrophils.

In conclusion, functional validation using iPSCs confirmed that these novel HYOU1 variants cause neutropenia in humans. Proteomic analysis of primary neutrophils suggests HYOU1's involvement in ER-to-Golgi intracellular trafficking. These findings underscore HYOU1's essential role in immune cell development and congenital neutropenia.