Comprehensive multi-omics analysis of the prognostic value and immune signature of NCF2 in pan-cancer and its relationship with acute myeloid leukemia.

BACKGROUND: The neutrophil cytoplasmic factor 2 (NCF2) gene encodes the p67phox protein, which has been implicated in the pathogenesis of several diseases. However, its specific role in tumorigenesis remains ambiguous. This study seeks to clarify the prognostic implications, immune profile, and therapeutic responses associated with NCF2 across different cancer types. METHODS: We conducted a comprehensive analysis using multi-omics data to investigate tissue-specific and single-cell specific expression, pan-cancer expression patterns, epigenetic modifications, the immune microenvironment, and therapeutic responses. Our study specifically examined NCF2-associated immune signatures, molecular mechanisms, and potential therapeutic targets in acute myeloid leukemia (AML). Additionally, we performed in vitro experiments to assess how NCF2 knockdown influences cell proliferation, apoptosis, and cell cycle dynamics in AML cell lines U937 and KG-1. RESULTS: NCF2 is dysregulated in more than two-thirds of cancer types, with elevated expression strongly correlating with poor prognosis in various cancers, including leukemia. Multifactorial Cox analysis has identified NCF2 as an independent prognostic factor for leukemia. Immunological studies have highlighted NCF2's impact on the tumor microenvironment, particularly affecting monocytes and macrophages. Furthermore, NCF2 expression closely correlates with responses to immunotherapy and chemotherapy. In vitro experiments demonstrate that NCF2 knockdown alters proliferation, apoptosis, and cell cycle dynamics of U937 cells and KG-1 cells. Notably, NCF2 is involved in regulating the differentiation of monocytes into macrophages. CONCLUSIONS: These findings highlight NCF2 as a promising pan-cancer biomarker that significantly impacts tumor microenvironment, therapeutic response, and is critically associated with cell cycle regulation, apoptosis and macrophage transformation in AML.

Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47 phox -deficient chronic granulomatous disease.

p47 phox -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47 phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67 phox , an NADPH oxidase subunit that closely interacts with p47 phox and is predominantly expressed in myeloid cells. This approach restored p47 phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47 phox -deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47 phox and p67 phox , ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.

Targeted serum proteome profiling reveals nicotinamide adenine dinucleotide phosphate (NADPH)-related biomarkers to discriminate linear IgA bullous disorder from dermatitis herpetiformis.

Linear IgA bullous dermatosis (LABD) and dermatitis herpetiformis (DH) represent the major subtypes of IgA mediated autoimmune bullous disorders. We sought to understand the disease etiology by using serum proteomics. We assessed 92 organ damage biomarkers in LABD, DH, and healthy controls using the Olink high-throughput proteomics. The positive proteomic serum biomarkers were used to correlate with clinical features and HLA type. Targeted proteomic analysis of IgA deposition bullous disorders vs. controls showed elevated biomarkers. Further clustering and enrichment analyses identified distinct clusters between LABD and DH, highlighting the involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Comparative analysis revealed biomarkers with distinction between LABD and DH and validated in the skin lesion. Finally, qualitative correlation analysis with DEPs suggested six biomarkers (NBN, NCF2, CAPG, FES, BID, and PXN) have better prognosis in DH patients. These findings provide potential biomarkers to differentiate the disease subtype of IgA deposition bullous disease.

Clinical and molecular significance of flow cytometric analysis for reactive oxygen species production and residual p67phox expression in p67phox-deficient chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by molecular defects in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. p67phox-CGD is an autosomal recessive CGD, which is caused by a defect in the cytosolic components of NADPH oxidase, p67phox, encoded by NCF2. We previously established a flow cytometric analysis for p67phox expression, which allows accurate assessment of residual protein expression in p67phox-CGD. We evaluated the correlation between oxidase function and p67phox expression, and assessed the relevancy to genotypes and clinical phenotypes in 11 patients with p67phox-CGD. Reactive oxygen species (ROS) production by granulocytes was evaluated using dihydrorhodamine-1,2,3 (DHR) assays. p67phox expression was evaluated in the monocyte population. DHR activity and p67phox expression were significantly correlated (r = 0.718, p < 0.0162). Additionally, DHR activity and p67phox expression were significantly higher in patients carrying one missense variant in combination with one nonsense or frameshift variant in the NCF2 gene than in patients with only null variants. The available clinical parameters of our patients (i.e., age at disease onset, number of infectious episodes, and each infection complication) were not linked with DHR activity or p67phox expression levels. In summary, our flow cytometric analysis revealed a significant correlation between residual ROS production and p67phox expression. More deleterious NCF2 genotypes were associated with lower levels of DHR activity and p67phox expression. DHR assays and protein expression analysis by using flow cytometry may be relevant strategies for predicting the genotypes of p67phox-CGD.

[Correlation study of NCF2 in chronic rhinosinusitis with nasal polyps].

Objective:After selecting NCF2 based on bioinformatics, clinical experiments were conducted to verify the expression of NCF2 in chronic rhinosinusitis with nasal polyps to study its correlation. Methods:The differentially expressed genes（DEGs） between CRSwNP and non-CRS patients were explored using the CRS-related dataset from the gene expression omnibus GEO database. The weighted gene co-expression network（WGCNA） was used for cluster analysis. The expression and cell distribution of NCF2 in the tissues were determined by single gene enrichment analysis（GSEA）, immune inflammatory infiltration analysis, and principal component（PCA） analysis. The expression degree of NCF2 in the tissues of the subjects was determined by immunohistochemistry, and the percentage of EOS in the peripheral blood of the subjects was detected and the correlation was analyzed. EOS in the tissues of the subjects were counted under a microscope and compared. Results:①The Venn diagram was obtained by crossing the module with the highest correlation between DEGs and WGCNA to determine the core gene NCF2. ②GSEA analysis showed that NCF2 was significantly related to the immunological processes such as allogeneic rejection and asthma. ③The area under the ROC curve was 1, indicating that NCF2 had diagnostic value for CRSwNP. ④NCF2 was highly expressed in nasal polyps, mainly distributed in monocytes and eosinophils. ⑤HE staining showed that the number of EOS in ECRSwNP tissues and the percentage of eosinophils in peripheral blood were higher than those in nonECRSwNP and control groups. ⑥The immunohistochemistry results showed that NCF2 was significantly expressed in the nasal polyps of ECRSwNP patients, which was higher than that in the nasal mucosa of nonECRSwNP group and control group. ⑦The expression of NCF2 in tissues was positively correlated with EOS count in ECRSwNP group and EOS expression in peripheral blood. Conclusion:The expression of NCF2 is increased in eosinophilic chronic rhinosinusitis with nasal polyps, and it is significantly correlated with the expression of eosinophils in peripheral blood and tissues, suggesting that NCF2 may be used as a basis for the intrinsic classification of ECRSwNP and a reference index for clinical diagnosis and treatment.

Novel biomarkers related to oxidative stress and immunity in chronic kidney disease.

Introduction: The incidence of chronic kidney disease (CKD) has been increasing in recent years, gradually becoming a global health crisis. Due to limited treatment options, novel molecular pathways are urgently required to advance the treatment and diagnosis of CKD. Materials and methods: The characteristics of differentially expressed genes (DEGs) in CKD patients were analyzed using Gene Expression Omnibus (GEO) database, and genes related to oxidative stress were retrieved from the Genecard database. Subsequently, a comprehensive approach was applied, including immune infiltration analysis, weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) network analysis, to identify hub genes among differentially expressed immune-related oxidative stress genes (DEIOSGs). Validation of hub genes was performed using an external data set, and diagnostic potential capability was evaluated through receiver operating curve (ROC) analysis. In animal experiments, the expression of hub genes in CKD was confirmed by inducing a CKD model through a 5/6 nephrectomy procedure. Finally, the relationship between these hub genes and clinical characteristics were assessed using the Nephroseq v5 database. Results: 29 DEIOSGs were identified by comprehensive bioinformatics analysis. PPI analysis screened the hub genes NCF2, S100A9, and SELL. ROC analysis demonstrated excellent diagnostic efficacy. Further validation from other databases and animal experiments confirmed a substantial upregulation in the expression of hub genes in CKD. Additionally, clinical correlation analysis established a clear link between hub gene expression and renal function deterioration. Conclusions: Our study confirms NCF2, S100A9, and SELL as diagnostic biomarkers associated with immune response and oxidative stress in CKD, suggesting their potential as novel targets for CKD diagnosis and treatment.

[Bacillus Calmette-Guérin infection and chronic granulomatous disease due to new pathogenic variants in the NCF2 gene in the Mayan ethnic group. Report of two cases.] / Infección por bacilo de Calmette-Guérin y enfermedad granulomatosa crónica por nuevas variantes patogénicas del gen NCF2 en la etnia maya. Reporte de dos casos.

INTRODUCTION: Chronic granulomatous disease (CGD) is an inborn error of immunity, characterized by abnormal susceptibility to bacterial and fungal infections and a lack of systemic inflammatory regulation. Pathogenic variants in the CYBB gene are transmitted in an X-linked pattern of inheritance; while the pathogenic variants present in the EROS, NCF1, NCF2, NCF4, or CYBA genes are transmitted with an autosomal recessive inheritance pattern. OBJETIVES: To describe the clinical, immunological, and genetic characteristics of two patients with CGD and BCG infection. METHODS: In peripheral blood neutrophils, H2O2 production and the expression of NADPH oxidase subunits were measured. Detection of pathogenic variants was by Sanger sequencing of the NCF2 gene. The clinical information was extracted from the records by the treating physicians. RESULTS: We present two male infants from two unrelated families of Mayan ethnicity, with CGD and BCG vaccine infection. Three different pathogenic variants in the NCF2 gene were identified; on the one hand, c.304 C>T (p.Arg102*) has already been reported, on the other hand, c.1369 A>T (p.Lys457*) and c.979 G>T (p.Gly327*) not reported. CONCLUSIONS: In patients with mycobacterial infection with BCG, we should suspect an inborn error of immunity, such as CGD. The diagnosis of CGD is made through the detection of a lack of radical oxygen species in neutrophils. The reported patients had pathogenic variants in the NCF2 gene, two of which have not been previously reported in the literature.

INTRODUCCIÓN: La enfermedad granulomatosa crónica (EGC) es un error innato de la inmunidad, se caracteriza por una susceptibilidad a padecer infecciones bacterianas y fúngicas y a una falta de regulación inflamatoria sistémica. Las variantes patogénicas en el gen CYBB se trasmiten con un patrón de herencia ligada al X; mientras que las variantes patogénicas presentes en los genes EROS, NCF1, NCF2, NCF4 o CYBA se trasmiten con un patrón de herencia autosómico recesivo. OBJETIVOS: Describir las características clínicas, inmunológicas y genéticas de dos pacientes con EGC e infección por BCG. MÉTODOS: En neutrófilos de sangre periférica se midió la producción de H2O2 y la expresión de las subunidades de la NADPH oxidasa. La detección de las variantes patogénicas fue por secuenciación Sanger del gen NCF2. La información clínica fue extraída de los expedientes por los médicos tratantes. RESULTADOS: Presentamos a dos lactantes masculinos de dos familias no relacionadas de la etnia maya, con EGC e infección por la vacuna de BCG. Se identificaron tres diferentes variantes patogénicas en el gen NCF2; por un lado, c.304 C>T (p.Arg102*) ya reportada, por otro lado, c.1369 A>T (p.Lys457*) y c.979 G>T (p.Gly327*) no reportadas. CONCLUSIONES: En pacientes con infección micobacteriana por BCG debemos sospechar en un error innato de la inmunidad, como la EGC. El diagnóstico de EGC se realiza a través de la detección de una falta de producción de radicales libres en los neutrófilos. Los pacientes reportados tuvieron variantes patogénicas en el gen NCF2, dos de ellas no han sido reportadas previamente en la literatura.

Identification of m6A regulator-mediated RNA methylation modification patterns and key immune-related genes involved in atrial fibrillation.

The role of m6A in the regulation of the immune microenvironment in atrial fibrillation (AF) remains unclear. This study systematically evaluated the RNA modification patterns mediated by differential m6A regulators in 62 AF samples, identified the pattern of immune cell infiltration in AF and identified several immune-related genes associated with AF. A total of six key differential m6A regulators between healthy subjects and AF patients were identified by the random forest classifier. Three distinct RNA modification patterns (m6A cluster-A, -B and -C) among AF samples were identified based on the expression of 6 key m6A regulators. Differential infiltrating immune cells and HALLMARKS signaling pathways between normal and AF samples as well as among samples with three distinct m6A modification patterns were identified. A total of 16 overlapping key genes were identified by weighted gene coexpression network analysis (WGCNA) combined with two machine learning methods. The expression levels of the NCF2 and HCST genes were different between controls and AF patient samples as well as among samples with the distinct m6A modification patterns. RT-qPCR also proved that the expression of NCF2 and HCST was significantly increased in AF patients compared with control participants. These results suggested that m6A modification plays a key role in the complexity and diversity of the immune microenvironment of AF. Immunotyping of patients with AF will help to develop more accurate immunotherapy strategies for those with a significant immune response. The NCF2 and HCST genes may be novel biomarkers for the accurate diagnosis and immunotherapy of AF.

Role of NCF2 as a potential prognostic factor and immune infiltration indicator in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related deaths globally. The tumor microenvironment (TME) plays a crucial role in the prognosis and treatment of HCC. Hence, it is important to exploit new biomarkers for survival surveillance and TME estimation of HCC. METHODS: HCC samples data was collected from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) database, and clinical samples were collected from our center. The TME of HCC were explored with ESTIMATE (Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data), ssGSEA (single sample Gene Sets Enrichment Analysis) and CIBERSORT algorithm. Differentially expressed genes were analyzed with functional enrichment analysis. Immunohistochemistry was implemented to validate the results. RESULTS: Based on TCGA database, we found that Neutrophil Cytosolic Factor 2 (NCF2) was significantly associated with the prognosis of HCC patients, involved in immune-related biological processes of HCC and closely associated with some types of immunocompetent cells. The survival analysis based on NCF2 expression assessed by immunohistochemistry also confirmed that NCF2-positive group had a shorter relapse free survival (RFS) and overall survival (OS) than NCF2-negative group. Multivariate Cox regression revealed NCF2 expression level and lymphovascular space invasion (LVSI) were independent risk factors for HCC patients. Receiver operating characteristic curves showed that the combination of NCF2 and LVSI had higher predictive efficacy on the 1-year RFS rate and 5-year OS rate than each of them alone. Besides, the expression level of NCF2 was positively associated with M0 and M2 macrophages infiltration. Furthermore, NCF2 expression was positively correlated with CSF1, IL4, IL10, CD206, CD163, CSF1R and TGFß1. CONCLUSION: We proposed that higher NCF2 expression predicted an adverse prognosis and more M2 macrophages infiltration in HCC patients.

Bisphenol A: Potential Factor of Miscarriage in Women in the Context of the Phenomenon of Neutrophil Extracellular Traps.

Humans are exposed to a number of environmental pollutants every day. Among them, endocrine disruptors are particularly harmful to human health. Bisphenol A (BPA) is a xenoestrogen that has been shown to disrupt the endocrine system and cause reproductive toxicity. In this study, we aimed to verify the potential relationship between BPA and miscarriage involving the formation of neutrophil extracellular traps (NETs). Blood samples were collected from healthy women and women who had miscarriage in the first trimester of pregnancy. The serum levels of cytoplasmic anti-PR3 antibody and perinuclear anti-MPO antibody were determined using an immunoenzymatic method. The concentrations of key proinflammatory proteins TNF-α and MCP-1, as well as NADPH oxidase subunits NOX1 and NCF2, were also measured in the serum samples. The serum concentration of BPA was determined using gas chromatography. The results showed that the concentrations of BPA were significantly elevated in the serum of women who had miscarriage compared to the control group, with the highest concentration found in the "NETs-positive" group. The levels of MCP-1 and TNF-α were significantly higher in the "NETs-positive" group compared to the "NETs-negative" and control group. The levels of NOX1 and NCF2 were also higher in the "NETs-positive" group compared to the "NETs-negative" group. The study showed that BPA could play a role in the course of miscarriage through the formation of NETs. The results indicate the need to limit the exposure of women planning pregnancy to xenoestrogens, including BPA.

The prognostic miR-532-5p-correlated ceRNA-mediated lipid droplet accumulation drives nodal metastasis of cervical cancer.

Introduction: The prognosis for cervical cancer (CC) patients with lymph node metastasis (LNM) is extremely poor. Lipid droplets (LDs) have a pivotal role in promoting tumor metastasis. The crosstalk mechanism between LDs and LNM modulated in CC remains largely unknown. Objectives: This study aimed to construct a miRNA-dependent progonostic model for CC patients and investigate whether miR-532-5p has a biological impact on LNM by regualting LDs accumulation. Methods: LASSO-Cox regression was applied to establish a prognostic prediction model. miR-532-5p had the lowest P-value in RNA expression (P < 0.001) and prognostic prediction (P < 0.0001) and was selected for further study. The functional role of the prognostic miR-532-5p-correlated competing endogenous RNA (ceRNA) network was investigated to clarify the crosstalk between LDs and LNM. The underlying mechanism was determined using site-directed mutagenesis, dual luciferase reporter assays, RNA immunoprecipitation assays, and rescue experiments. A xenograft LNM model was established to evaluate the effect of miR-532-5p and orlistat combination therapy on tumor growth and LNM. Results: A novel 5-miRNAs prognostic signature was constructed to better predict the prognosis of CC patient. Further study demonstrated that miR-532-5p inhibited epithelial-mesenchymal transition and lymphangiogenesis by regulating LDs accumulation. Interestingly, we also found that LDs accumulation promoted cell metastasis in vitro. Mechanistically, we demonstrated a miR-532-5p-correlated ceRNA network in which LINC01410 was bound directly to miR-532-5p and effectively functioned as miR-532-5p sponge to disinhibit its target gene-fatty acid synthase (FASN). Combined therapy with miR-532-5p and FASN inhibitor-orlistat further inhibited tumor growth and LNM in vivo. Conclusion: Our findings highlight a LD accumulation-dependent mechanism of miR-532-5p-modulated LNM and support treatment with miR-532-5p/orlistat as novel strategy for treating patients with LNM in CC.

Neutrophil cytosolic factor 2 (NCF2) gene polymorphism is associated with juvenile-onset systemic lupus erythematosus, but probably not with other autoimmune rheumatic diseases in children.

BACKGROUND: Genetic variations of neutrophil cytosolic factor 2 (NCF2), a subunit of NADPH oxidase, are usually associated with chronic granulomatous disease, and their relationship with autoimmune disorders through the defective NADPH oxidase function during phagocytosis is suggested. Our study aimed to explore whether there is an association between the non-synonymous single nucleotide polymorphism in the NCF2 gene (rs17849502, NC_000001.11:g.183563445G>T) and the development of juvenile autoimmune rheumatic diseases. METHODS: In order to test this hypothesis, we conducted a pilot case-control study. In total, 709 children and adolescents, all Belarusians, were involved in the study including patients with juvenile-onset systemic lupus erythematosus (JSLE), juvenile idiopathic arthritis (JIA), Kawasaki disease (KD), and subjects without autoimmune and inflammatory diseases as the clinical control, as well as health newborns as the population control. Real-time polymerase chain reaction was used for genotyping. RESULTS: The minor T allele of NCF2 occurred most frequently in patients with JSLE (OR = 2.60, 95% CI = 1.18-5.73, p = 0.023 as compared to the clinical control). In groups with JIA and KD, its frequency did not differ from the control. The TT genotype was only observed in 5.7% of patients with JSLE (p = 0.007), but not in other groups. CONCLUSION: Therefore, our study suggested that NCF2 rs17849502 polymorphism is a potential genetic risk factor for JSLE, while it is probably not for such autoimmune rheumatic diseases as JIA or KD.

Utility of Immunohistochemistry and Immunofluorescence in Determining the Pathogenic Variants of Chronic Granulomatous Disease.

BACKGROUND: Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder of phagocytes due to defects in any of the five subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. An initial diagnosis of CGD is made by flow cytometry-based dihydrorhodamine assay or nitro blue tetrazolium test, which is further confirmed by molecular assays. Expression of five subunits of NADPH oxidase components by either flow cytometric or western blot analysis provides clues toward the potential gene targets which are subsequently confirmed by various genetic assays. Immunohistochemistry (IHC) and immunofluorescence (IF) have never been earlier used to determine the expression of different subunits of NADPH oxidase system. We evaluated the utility of IHC and IF in determining the underlying pathogenic variants of CGD. MATERIALS AND METHODS: Twelve genetically confirmed cases of CGD, comprising of biopsy specimens (n = 6), tissue blocks from autopsy cases (n = 3), and cellblocks of cell pellet prepared from peripheral blood (n = 4) were included. IHC for p67phox and p47phox subunits and IF for cytochrome b558 were performed. RESULTS: All 4 cases with pathogenic variation of NCF2 gene showed loss of expression for p67phox subunit. Two cases with pathogenic variation of NCF1 gene showed loss of expression for p47phox subunit. Five cases, except a single case with CYBB gene pathogenic variation, showed loss of expression for cytochrome b558 on IF. Thus, loss of expression consistently matched with the underlying genetic defects assessed by sequencing. CONCLUSIONS: Our results confirm our hypothesis that IHC and IF are two rapid, economical, pathologist-friendly techniques providing pertinent information regarding the underlying pathogenic variants and such immuno-analysis can be easily performed on the tissue.

Altered NCF2, NOX2 mRNA Expression Levels in Peripheral Blood Mononuclear Cells of Pulmonary Tuberculosis Patients.

BACKGROUND: Reactive oxygen species (ROS) generated by NADPH oxidase has a pivotal role in the nonspecific innate immune response to invading microorganisms including M. tuberculosis (MTB). NCF2 and NOX2 were considered as important functional subunits of NADPH oxidase complex; hence, this study aimed to evaluate the NCF2, NOX2 mRNA expressions in PBMC of pulmonary tuberculosis (PTB) patients. METHODS: A total of 79 PTB patients and 73 controls were included in our study. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to measure the NCF2, NOX2 mRNA levels, and receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic value of NCF2, NOX2 in PTB patients. RESULTS: When compared to controls, the NCF2, NOX2 mRNA levels were significantly increased in PBMC from PTB patients (P < 0.001). However, the NCF2, NOX2 mRNA levels were not associated with major clinical and laboratory data of PTB patients. Area under curve (AUC) of ROC curve analysis for NCF2 and NOX2 were 0.686 (95% CI: 0.601, 0.770) and 0.705 (95% CI: 0.623, 0.787), respectively. CONCLUSION: Altered NCF2, NOX2 mRNA levels in PTB patients implied that these genes might play roles in PTB, and their expression levels might be potential biomarkers for the diagnosis of PTB.

Novel NCF2 Mutation Causing Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder caused by defects in the NADPH oxidase complex. Mutations in NCF2 encoding the cytosolic factor p67phox result in autosomal recessive CGD. We describe three patients with a novel c.855G>C NCF2 mutation presenting with diverse clinical phenotype. Two siblings were heterozygous for the novel mutation and for a previously described exon 8-9 duplication, while a third unrelated patient was homozygous for the novel mutation. Mutation pathogenicity was confirmed by abnormal DHR123 assay and absent p67phox production and by sequencing of cDNA which showed abnormal RNA splicing. Clinically, the homozygous patient presented with suspected early onset interstitial lung disease and NCF2 mutation was found on genetic testing performed in search for surfactant-related defects. The two siblings also had variable presentation with one having history of severe pneumonia, lymphadenitis, and recurrent skin abscesses and the other presenting in his 30s with discoid lupus erythematosus and without significant infectious history. We therefore identified a novel pathogenic NCF2 mutation causing diverse and unusual clinical phenotype.

A novel variant in the neutrophil cytosolic factor 2 (NCF2) gene results in severe disseminated BCG infectious disease: A clinical report and literature review.

BACKGROUND: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder (PID) affecting NADPH oxidase activity. The rarest form of the disease is considered to be caused by NCF2 gene bi-allelic variant. Here, we report the clinical and molecular characterization of a patient presenting with early-onset severe disease due to bi-allelic NCF2 variant. METHODS: Gene mutational analysis was performed by whole-exome and Sanger sequencing. RESULTS: The patient presented with a history of fever and rash since the age of 1 month, followed by destructive osteomyelitis and necrotizing lymphadenopathy. The patient received the Bacillus Calmette-Guérin (BCG) vaccine at birth; she was subsequently diagnosed with disseminated BCG infection. Whole-exome sequencing identified a private (unreported) homozygous variant in NCF2 (c.290C > A) that results in a nonconservative change, p.Ala97Asp, in the p67phox protein. The variant is located in the third helix of the TRP domain, which is crucial for the binding of GTPase RAC2 to the NADPH oxidase complex. CONCLUSION: We identified a novel NCF2 variant located in the region interacting with RAC2 that is linked to a severe and early CGD phenotype in the setting of disseminated BCG infection. Our findings support postponing BCG vaccination until 6-12 months of age and after PID assessment.

Low-Dose Pioglitazone does not Increase ROS Production in Chronic Granulomatous Disease Patients with Severe Infection.

PURPOSE: We sought to further investigate the efficacy and safety of pioglitazone for chronic granulomatous disease (CGD) patients with severe infection. METHODS: CGD patients with severe infection were enrolled and treated with pioglitazone for 90 days. The degree of improvement in infection and the changes of dihydrorhodamine-123 (DHR) were used to evaluate the efficacy of pioglitazone. The adverse reaction of pioglitazone was also investigated. RESULTS: We planned to enroll 30 patients at first in the study. However, the study was terminated due to negative results from all 3 enrolled patients. The 3 patients were diagnosed with CGD by clinical characteristics, DHR analysis, and genetics analysis. Mutations were CYBB (c.177C>A; p.C59X) in P1, CYBB (c.1498G>T; p.D500Y) in P2, and NCF2 (c.137T>G; p.M46R) in P3, respectively. The age of onset of the 3 patients was within 2 years after birth. The most common sites of infection were lung, lymph node, skin, and soft tissue, which were experienced in all 3 patients. The age of administration with pioglitazone was 5.2 years, 16 years and 11.1 years, respectively. The 3 patients experienced no improvement in severity of infection and stimulation index of the DHR did not also improve after receiving pioglitazone 10, 45 and 90 days, respectively. No drug-related adverse reaction was found during the period of pioglitazone. CONCLUSIONS: Low dose of pioglitazone did not improve the severity of infection and production of ROS in CGD patients with severe infection.

High expression of neutrophil cytosolic factor 2 (NCF2) is associated with aggressive features and poor prognosis of esophageal squamous cell carcinoma.

BACKGROUND: In the development of several human cancers, it has been established that neutrophil cytosolic factor 2 (NCF2) plays a major part. Therefore, possible functions of NCF2 in ESCC are investigated in this paper. METHODS: The mRNA/protein expression of NCF-2 in ESCC cell lines and tissues were found based on quantitative real-time reverse transcription PCR (qRT-PCR), western blotting, and immunohistochemistry (IHC). A large cohort consisting of 194 postoperative ESCC samples was used for IHC. These data were analyzed based on Chi-square test, Kaplan-Meier analysis, and Cox regression modelling. For the purpose of confirming its role in ESCC cells, we used short hairpin RNA (ShRNA) interfering method to suppress endogenous NCF2 expression. RESULTS: NCF2 was significantly up-regulated for in ESCC tissues and cell lines in at mRNA and protein levels; and NCF-2 expression was absent for all normal esophageal epithelium detected by IHC. Furthermore, the knockdown of NCF-2 compromised the proliferation and invasion of ESCC cells in vitro. CONCLUSION: Positive NCF2 expression in ESCC may facilitate an aggressive phenotype. This may be an independent biomarker in ESCC.

Source of Dietary Fat in Pig Diet Affects Adipose Expression of Genes Related to Cancer, Cardiovascular, and Neurodegenerative Diseases.

It has been known for many years that excessive consumption of saturated fats has proatherogenic properties, contrary to unsaturated fats. However, the molecular mechanism covering these effects is not fully understood. In this paper, we aimed to identify differentially expressed genes (DEGs) using RNA-sequencing, following feeding pigs with different sources of fat. After comparison of adipose samples from three dietary groups (rapeseed oil (n = 6), beef tallow (n = 5), coconut oil (n = 5)), we identified 29 DEGs (adjusted p-value < 0.05, fold change > 1.3) between beef tallow and rapeseed oil and 2 genes between coconut oil and rapeseed oil groups. No differentially expressed genes were observed between coconut oil and beef tallow groups. Almost all 29 DEGs between rapeseed oil and beef tallow groups are connected to neurodegenerative, cardiovascular diseases, or cancer (e.g., PLAU, CYBB, NCF2, ZNF217, CHAC1, CTCFL). Functional analysis of these genes revealed that they are associated with fluid shear stress response, complement and coagulation cascade, ROS signaling, neurogenesis, and regulation of protein binding and protein catabolic processes. Furthermore, gene set enrichment analysis (GSEA) of the whole datasets from all three comparisons suggests that both beef tallow and coconut oil may trigger changes in the expression level of genes crucial in the pathogenesis of civilization diseases.