As the diverse functions of neutrophils continue to be uncovered, elucidating the molecular mechanisms that regulate their differentiation, development, and apoptosis has become crucial for overcoming limitations in the treatment of neutrophil-related diseases. Hematopoietic cell-specific protein 1-associated protein X 1 (HAX1), encoded by the primary pathogenic gene of autosomal recessive severe congenital neutropenia, serves as a key target for in-depth exploration of neutrophil function. In the Hax1 myeloid knockout C57BL/6J mice and stably transduced HL-60 cells with HAX1 knockdown that we constructed, our results showed that the differentiation of granulocyte-monocyte precursor cells (GMPs) and the maturation of neutrophils were inhibited, significantly reducing the proportion of myeloid cells and neutrophils in both bone marrow and peripheral blood. In addition, HAX1 deletion disrupted mitochondrial structure and mitochondrial membrane potential in neutrophils and increased the protein levels of B-cell lymphoma 2 (BCL-2) family members and cleaved Caspase-9. Through RNA sequencing and mRNA validation, we further demonstrated that HAX1 regulates neutrophil apoptosis and maturation via the mitochondrial-mediated classical apoptotic pathway and toll-like receptor 2 (TLR2)-mediated purine-rich box 1 (PU.1) signaling. This study elucidated the critical role of HAX1 in neutrophil differentiation, maturation, and apoptosis, providing new targets for research into neutrophil-related diseases. HAX1 regulates neutrophil apoptosis through mitochondrial dependent pathway and multiple TLR2 mediated pathways, affecting neutrophil maturation by regulating PU.1 levels via TLR2.

Autosomal recessive severe congenital neutropenia (SCN) has been associated with homozygous variants in the HAX1 gene. The aim of this cross-sectional study was to evaluate the gonadal function and pubertal development in pediatric patients with SCN due to HAX1 gene variant (HAX1-SCN). Forty-five patients, including 24 females (median age 11.3 [1.5-31] years, 13 pubertal, 11 prepubertal), and 21 males (median age 9.5 (3-18.8) years, 7 pubertal, 14 prepubertal), followed in seven centers, were included. POI is defined as a menstrual disturbance with increased follicle-stimulating hormone (FSH) and low anti-Mullerian hormone (AMH). We classified prepubertal female patients as impending POI when they had low AMH and high FSH values, indicating impaired ovarian function. A homozygous single nucleotide insertion (position 130-131insA) leading to a premature stop codon; p.Trp44*(c.132G>A) variant in HAX1 gene was detected in 42 (93.3%) affected individuals. Other homozygous variants were p.Arg86*(c.256C>T) and p.Glu60Aspfs*25(c.180delA). We detected elevated serum FSH levels in 10/11 (90.9%) of prepubertal female patients, supporting the diagnosis of impending POI, and in 12/13 (92.3%) of pubertal female patients, classifying them as POI. All female patients had low AMH levels. Male patients did not exhibit gonadal insufficiency. This is the first and largest case series covering early childhood to evaluate patients with HAX1-SCN for gonadal function. It has been observed that pubertal females develop POI, prepubertal females are at increased risk for gonadal failure, and male patients are not affected. Our results suggest that HAX1 has an important role in ovarian maturation and/or function.

Severe congenital neutropenia presents with recurrent infections early in life as a result of arrested granulopoiesis. Multiple genetic defects are known to block granulocyte differentiation; however, a genetic cause remains unknown in approximately 40% of cases. We aimed to characterize a patient with severe congenital neutropenia and syndromic features without a genetic diagnosis. Whole exome sequencing results were validated using flow cytometry, Western blotting, coimmunoprecipitation, quantitative PCR, cell cycle and proliferation analysis of lymphocytes and fibroblasts and granulocytic differentiation of primary CD34+ and HL-60 cells. We identified a homozygous missense mutation in DBF4 in a patient with mild extra-uterine growth retardation, facial dysmorphism and severe congenital neutropenia. DBF4 is the regulatory subunit of the CDC7 kinase, together known as DBF4-dependent kinase (DDK), the complex essential for DNA replication initiation. The DBF4 variant demonstrated impaired ability to bind CDC7, resulting in decreased DDK-mediated phosphorylation, defective S-phase entry and progression and impaired differentiation of granulocytes associated with activation of the p53-p21 pathway. The introduction of wild-type DBF4 into patient CD34+ cells rescued the promyelocyte differentiation arrest. Hypomorphic DBF4 mutation causes autosomal-recessive severe congenital neutropenia with syndromic features.